bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 125: Perturbation of Mitochondrial Calcium Uniporter Promotes Cardiac Oxidative Stress and Autophagy During Heart Failure
Authors: Rajan Sudarsan , Shanmughapriya Santhanam , Tomar Dhanendra , Dong Zhiwei , Cheung Joseph Y , Stathopulos Peter B , Muniswamy Madesh
Published: July 2021

Mitochondrial calcium ([Ca
2+
]
m
) is essential for cardiomyocyte viability, and aberration of [Ca
2+
]
m
is known to elicit multiple cardiac stress conditions associated with ATP depletion, reactive oxygen species, and mitochondrial permeability transition pore opening, all of which can lead to metabolic stress and the loss of dysfunctional mitochondria by aberrant autophagy. Elucidating the regulatory role of
m
itochondrial
c
alcium
u
niporter (MCU)-mediated [Ca
2+
]
m
in modulating cardiac mitochondrial bioenergetics and autophagy has high significance and clinical impact for many pathophysiological processes. [Ca
2+
]
m
is exquisitely controlled by the inner mitochondrial membrane uniporter, transporters, regulators and exchangers including MCU, MCUR1, EMRE, MICU1, MICU2 and LETM1. Our recently published findings revealed that Mitochondrial Ca
2+
Uniporter Regulator 1 (MCUR1) serves as a scaffold factor for uniporter complex assembly. We found that deletion of MCUR1 impaired [Ca
2+
]
m
uptake, mitochondrial Ca
2+
current (
I
MCU
) and mitochondrial bioenergetics and is associated with increased autophagy. Our new findings indicate that the impairment of [Ca
2+
]
m
uptake exacerbated autophagy following ischemia-reperfusion (I/R) injury. In support of our mouse model, human failing hearts show that MCUR1 protein levels are markedly decreased and autophagy markers are increased, demonstrating a crucial link between [Ca
2+
]
m
uptake and autophagy during heart failure. Additionally, our results reveal that either oxidation or disruption of human MCU Cys-97 (in mouse Cys-96; gain-of-function MCU
C96A
mutant) produces a conformational change within the N terminal β-grasp fold of MCU which promotes higher-order MCU complex assembly and increased
I
MCU
activity and mitochondrial ROS levels. The results of our studies using a novel cardiac-specific MCUR1-KO model and a constitutively active global MCU
C96A
KI mouse model (CRISPR-Cas9 genome edited) elucidate the regulatory role of [Ca
2+
]
m
in cardiac bioenergetics and autophagy during oxidative stress and myocardial infarction. Thus, targeting assembly and the activity of MCU complex will offer a new potential therapeutic target in the treatment of cardiomyopathy and heart failure.
Abstract 141: Class I Histone Deacetylases Localize to Cardiac Myocyte Mitochondria and Contribute to Ischemia Reperfusion Injury
Authors: Herr Daniel J , Baarine Mauhamad , Aune Sverre , Beeson Craig C , Ball Lauren E , Menick Donald R
Published: July 2021

Approximately half of the damage done to the heart by a myocardial infarction occurs during reperfusion of the ischemic region while the patient is in the care of the treatment team. While many different adjuvant treatments have been explored in an attempt to attenuate this ischemia-reperfusion (I/R) injury, little progress has been made in translating novel therapies to the clinic. Recently, it was discovered that epigenetic enzymes contribute to reperfusion-induced damage, but little is known about the exact mechanism by which they exacerbate I/R injury. Previously, we have shown that class I histone deacetylase (HDACs) activity acutely exacerbates I/R injury, and that inhibition of class I HDACs with MS-275 (entinostat) preserves left-ventricular (LV) function and substantially reduces the area of infarcted tissue in isolated rat hearts subjected to ischemia-reperfusion (IR) injury. Notably, this protective effect occurs whether MS-275 is given as a pretreatment or during the reperfusion phase alone. Given the acute nature of this protective effect, we hypothesized that class I HDACs mediate reperfusion injury by modulating the acetylation state of non-histone proteins in signaling cascades that are essential to cell survival. To examine this, hearts from male Sprague-Dawley rats were subjected to
ex vivo
I/R injury +/- class I HDAC inhibition during reperfusion. We then performed mass spectrometry to analyze the changes in the acetylome between sham and I/R groups with and without class I HDAC inhibition. Unexpectedly, mass spectrometry analysis revealed significant changes in the acetylation state of multiple mitochondrial enzymes. Further biochemical studies show that class I HDACs localize to cardiac mitochondria and may directly modulate mitochondrial acetylation. Interestingly, these effects are correlated with a reduction in the mitochondrial respiratory capacity and mitochondrial oxidative stress during reperfusion. This study is the first to identify a class I HDAC that localizes to the mitochondria and emphasizes the importance of exploring class I HDAC inhibitors for protection against ischemia-reperfusion injury.
Abstract 151: The Cardioprotective Effect of HIF-1α/miR-21/PDCD4 Pathway During Transplanted Heart Cold Storage
Authors: Zhu Liang , Mu Jingzhou , Qu Chang , Wu Yunhong , Li Shuzhuang , Ma Tonghui
Published: July 2021

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that plays a major role under hypoxia condition. Cold storage during heart transplantation causes the donor heart long-term hypoxia. There are some evidence indicating a conceivable HIF-1α/miR-21/PDCD4 pathway. We assessed the hypothesis that Hypoxia-inducible factor -1α (HIF-1α) has a cardioprotective effect during donor heart cold storage, by making the miR-21 upregulate reduce the expression of PDCD4. We establish the rats heart cold storage model and stratified into 6-hour groups from 0 to 24 hours. Western blot and RT-qPCR were performed to detected the expression of HIF-1α, miR-21, PDCD4 and PTEN. After cold storage the expression of HIF-1α increased from 0 to 6 hours, then gradually decreased but the expression level was relatively higher compared to control group. The miR-21 was upregulated from 0 to 12 hous then downregulated. The mRNA expression of PDCD4 was upregulated gradually, but the protein expression was significantly downregulated in the 12 hour then continued to upregulate. Interestingly, the expression level of miR-21 was highest in the 12 hour, which indicated miR-21 could inhibite the PDCD4. And we subsequently detected the mRNA of PTEN which can inhibit the HIF-1α and be inhibited by miR-21.The expression of PTEN was also significantly downregulated in the 12 hour. In conclusion, there is possible interaction between HIF-1α and miR-21, and the conceivable HIF-1α/miR-21/PDCD4 pathway play a protective role in cold storage of heart.

Key words:
Heart Transplantation, Cold storage, miR-21, HIF-1α, PDCD4
Abstract 158: A Tether Containing a UBX Domain (TUG) Mediates Glucose Transporter Translocation in the Ischemic Heart
Authors: Li Ji , Ma Yina , Bogan Jonathan
Published: July 2021

Introduction:
The adaptive metabolic regulation of glucose and fatty acid in the heart plays a critical role in limiting cardiac damage caused by ischemia and reperfusion (I/R). TUG (tether containing a UBX domain, for GLUT4) can be cleaved to mobilize glucose transporter GLUT4 from intracellular vesicles to the cell surface in skeletal muscle and adipose in response to insulin stimulation. The energy sensor AMP-activated protein kinase (AMPK) plays an important cardioprotective role in response to ischemic insults by modulating GLUT4 translocation.

Hypothesis:
TUG is one of the downstream targets of AMPK in the heart. TUG could be phosphorylated by ischemic AMPK and cleaved to dissociate with GLUT4 and increase GLUT4 translocation in the ischemic heart.

Methods:
In vivo
regional ischemia by ligation of left anterior coronary artery and
ex vivo
isolated mouse heart perfusion Langendorff system were used to test the hypothesis.

Results:
Antithrombin (AT) is an endogenous AMPK agonist in the heart and used to define the role of TUG in regulating GLUT4 trafficking during ischemia and reperfusion in the heart. AT showed its cardioprotective function through recovering cardiac pumping function and activating AMPK. The results showed that AMPK activation by AT treatment was through LKB1 and Sesn2 complex. Furthermore, the
ex vivo
heart perfusion data demonstrated that AT administration significantly increase GLUT4 translocation, glucose uptake, glycolysis and glucose oxidation during ischemia and reperfusion (p<0.05
vs
. vehicle). Moreover, AT treatment increased abundance of a TUG cleavage product (42 KD) in response to I/R. The TUG protein was clearly phosphorylated by activated AMPK in HL-1 cardiomyocytes. The
in vivo
myocardial ischemia results demonstrated that ischemic AMPK activation triggers TUG cleavage and significantly increases GLUT4 translocation to the cell surface. Moreover, an augmented interaction between AMPK and TUG was observed during ischemia.

Conclusions:
Cardiac AMPK activation stimulates TUG cleavage and causes the dissociation between TUG and GLUT4 in the intracellular vesicles. TUG is a critical mediator that modulates cardiac GLUT4 translocation to cell surface and enhances glucose uptake by AMPK signaling pathway.
Abstract 223: Troponin I Tyrosine Phosphorylation Modulation of Cardiac Function
Authors: Brundage Elizabeth A , Agatisa-Boyle Brendan , Shettigar Vikram , Chung Jae-Hoon , Qian Ziqing , Salhi Hussam E , Janssen Paul M , Pei Dehua , Davis Jonathan P , Ziolo Mark T , Biesiadecki Brandon J
Published: July 2021

Heart failure results in depressed contraction and slowed relaxation, both of which limit heart function and contribute to the progression of heart disease. Currently there is no chronic therapy to accelerate relaxation and reverse the diastolic dysfunction present in heart failure. Myocardial relaxation is regulated by serine/threonine phosphorylation of key regulatory proteins. Tyrosine (Tyr) specific kinases are expressed in the heart but the Tyr phosphorylation of regulatory proteins to modulate heart function has not been demonstrated. To investigate the effects of Tyr kinase phosphorylation on cardiac contraction we employed a novel cell penetrating peptide to deliver a direct Tyr kinase activator into isolated adult myocytes. Results demonstrate Tyr kinases activation increases Tyr phosphorylation of the regulatory protein troponin I (TnI) at Tyr26. We have demonstrated that TnI Tyr26 phosphorylation is beneficial to cardiac health by decreasing calcium sensitivity and accelerating myofilament deactivation (key determinants in accelerating myocardial relaxation) and that TnI Tyr26 phosphorylation undergoes functional integration with TnI Ser23/24 resulting in further accelerated calcium dissociation (accelerated relaxation) without further decreased calcium sensitivity (no further depression of contraction). We now demonstrate TnI Tyr26 also undergoes novel signaling integration with TnI Ser23/24 phosphorylation increasing the rate of Tyr kinase mediated Tyr26 phosphorylation. For the first time we demonstrate tyrosine kinase phosphorylation of TnI at Tyr26 modulates cardiac function resulting in accelerated relaxation. Increasing TnI Tyr26 phosphorylation may therefore serve as a novel targeted mechanism for future therapeutic development to accelerate depressed myocardial relaxation and improve diastolic dysfunction in heart failure.
Abstract 247: The Endothelial Transcription Factor GATA2 Prevents the Development of Heart Failure During Pressure Overload Through Suppression of the Two Secreted Long Non-coding RNAs GADLOR1 and 2
Authors: Froese Natali , Szaroszyk Malgorzata , Kattih Badder , Korf-Klingebiel Mortimer , Camper Sally A. , Molkentin Jeffery D , Wollert Kai C , Bauersachs Johann , Heineke Jörg
Published: July 2021

Capillary endothelial cells influence myocardial growth and function during pathological stress by releasing paracrine factors. We found that the transcription factor GATA2 is suppressed in cardiac endothelial cells by mechanical stimuli, and that GATA2 is downregulated in human failing hearts. To investigate the functional consequence of reduced endothelial GATA2 expression, we exposed endothelial cell specific, inducible GATA2 knock-out (G2-EC-KO) or wild-type (WT) mice to pressure overload through transverse aortic constriction (TAC). G2-EC-KO mice developed aggravated heart failure after TAC, but not enhanced fibrosis or capillary rarefaction. Investigation of stress signaling pathways revealed a prominent activation of p38 MAP kinases and Akt in cardiomyocytes after TAC only in WT mice, but not in G2-EC-KO mice, which in addition exerted increased calcineurin/NFAT activation. Transcriptional profiling revealed a strong upregulation of two distinct previously unknown long non-coding (lnc) RNAs in cardiac endothelial cells from G2-EC-KO mice, which we termed GADLOR1 and GADLOR2 and which were also induced in human failing hearts. Both RNAs were also induced in cultured cardiac endothelial cells in vitro after ablation of GATA2 and were found to be secreted within extracellular vesicles. Isolated cardiomyocytes incubated with extracellular vesicles from GATA2 depleted endothelial cells efficiently incorporated GADLOR1 and 2. Uptake of GADLORs by cardiomyocytes led to a profound reduction of p38 MAPK and Akt activation. Proteomic screening revealed that GADLOR1/2 bind the Ras-like protein TC21 in cardiomyocytes and block downstream signaling by preventing TC21 binding to its target PI3K. Application of both lncRNAs to the mouse myocardium by exosomal gene-transfer triggered aggravated cardiac dysfunction and disturbed stress signaling, while in turn inhibition of GADLOR1/2 by specific GapmeRs rescued stress signaling and cardiac dysfunction in G2-EC-KO mice during TAC. In summary, GATA2 protects the heart during pressure overload by suppressing the endothelial release of two long non-coding RNAs, which interfere with stress signaling in cardiomyocytes and ultimately induce heart failure.
Abstract 272: The Orphan Receptor GPRC5B Increases Pro-Inflammatory Signaling in Neonatal Rat Cardiac Fibroblasts and Cardiac MMP-9-expression
in vivo
Authors: von Samson-Himmelstjerna Friedrich A , Freundt Greta , Frey Norbert , Hippe Hans-Joerg
Published: July 2021

Background:
Inflammatory processes driven by cardiac fibroblasts (CF) play a major role in cardiac fibrosis and eventually diastolic dysfunction leading to heart failure. GPRC5B, a novel G protein-coupled orphan receptor, is involved in inflammatory pathways in adipocytes and endogenously expressed in CF.

Hypothesis:
We assessed the impact of GPRC5B on inflammatory and fibrotic pathways in isolated neonatal rat CF and mouse heart.

Methods & Results:
We show that 48h-stimulation of CF with Tumor Necrosis Factor α (TNFα, 50ng/ml) or Lipopolysaccharides (LPS, 100ng/ml) leads to a significant up-regulation of GPRC5B protein levels compared to unstimulated cells
(TNFα +74%, p<0.01; LPS +111%, p<0.001, n=8)
. This is confirmed by evaluation of mRNA levels after 12h-stimulation (
TNFα +69%, p<0.01; LPS +61%, p<0.01, n=9)
. Similarly, a mechanical stretch of 18% of CF length for 24h increases GPRC5B-mRNA by 83%
(p<0.01, n=6).

Adenoviral overexpression of GPRC5B results in an increased TNFα- (
+84%, p<0.01)
, Interleukin 1ß- (IL1ß,
+56%, p<0.05
), Interleukin 6- (IL6,
+32%, p<0.05
) and Matrix-Metalloproteinase 9- (MMP9,
+133%, p<0.01
) mRNA production in CF
(
AdLacZ vs. AdGPRC5B,
n=9)
. After an additional 24h-stimulation with LPS (10ng/ml), IL1ß-, IL6- and MMP9-mRNA increases as compared to AdLacZ-infected control cells
(
AdLacZ+LPS vs. AdGPRC5B+LPS
: IL1ß +114%, p<0.01; IL6 +113%, p<0.05; MMP9 +195%, p<0.01; n=8).

Conversely, siRNA mediated knockdown to 25% of endogenous protein levels followed by 24h-stimulation with LPS (10ng/ml) lowers significantly the expression of TNFα-, IL1ß- and IL6-mRNA when compared to control-transfected cells
(siNeg+LPS vs. siGPRC5B+LPS: TNFα -28%, p<0.05; IL1ß -30%, p<0.05; MMP9 -34%, p<0.05; n=8).

These findings are supported by Western Blot analysis of heart tissue from GPRC5B-transgenic mice revealing an increased protein expression of MMP9 in contrast to wild type mice
(WT vs. GPRC5B-TG: MMP9 +60%, p<0.001; n=10).

Conclusion:
GPRC5B is up-regulated in cardiac models of inflammation and mechanical stress. GPRC5B modulates cellular inflammatory response by increasing the levels of key cytokines in CF and of MMP9 expression in mouse heart pointing to a role in myocardial inflammation and cardiac remodeling.
Abstract 288: Use of hiPSC-Derived Cardiomyocytes to Explore Functional Cardiotoxicities of Direct Acting Antiviral Nucleoside Based Structures
Authors: Imredy John P , Balasubramanian Bharathi , Lagrutta Armando , Olsen David B , Bogen Stephane , Bulger Paul G , Vu Heather , Xu Qiuwei
Published: July 2021

Nucleoside-derived structures constitute important Direct-Acting Antivirals (DAAs) for Hepatitis C Virus (HCV) treatment. BMS-986094 (BMS-094: a 2’-C-modified guanosine (G) derivative prodrug) was discontinued in phase II due to heart/kidney failure and cardiomyopathy, whereas sofosbuvir (a 2’-C-modified uridine prodrug) is an effective HCV DAA. Given the importance of purine nucleosides in cardiac signaling, we investigated BMS-094 in the human induced pluripotent stem cell derived cardiomyocyte (hiPSC-CM) model (iCells, CDI Fujifilm). Beating myocyte monolayer impedance signals were monituored for up to 7 days in the RTCA Cardio platform (ACEA-Biosystems). BMS-094 was acutely myotoxic at high concentrations (≥ 17 μM), as evidenced by cessation of myocyte beating and loss of baseline impedance or ‘Cell Index’ (C.I.), a measure of cell adherence, within hours. On prolonged exposure to BMS-094 at concentrations not associated with loss of C.I. (< 1 μM), we observed progressive, dose-dependent decreases in impedance amplitude (myocyte contractility) which were accompanied by increases in the spontaneous myocyte beating rate. The same beating phenotype could be elicited over a multi-day exposure duration by extracellular application of the BMS-094 core nucleoside (2’-C-methyl-guanosine) alone, but not by unmodified G. Loss of contractility with the G derivatives was accompanied by a decrease of the Ca
2+
transient amplitude in a Ca
2+
influx fluorescence assay (FDSS μCell, Hamamatsu). We investigated the impact of purine or pyrimidine base substitutions in the 2’-CMe-modified nucleoside: 2’-CMe-adenosine, -cytidine, -G, and -uridine. The uridine analog had the most limited effects on hiPSC-CMs beating parameters. The effect of 2’-CMe-cytidine on beating rate and impedance amplitude was similar to that of the G derivative, including a progressive multi-day onset. In contrast, 2’-CMe-adenosine slowed beating acutely, and led to loss of C.I. at higher concentrations. These results demonstrate the varied effects on CM function of base substitutions in antiviral nucleoside chemistry and support the utility of hiPSC-CMs as a model for screening
in-vitro
the potential cardiotoxicities of nucleoside derivative drugs.
Abstract 289: Improved Disease Modeling Reveals 9p21 Risk Allele Regulates Connexins to Induce Arrhythmic Phenotypes
Authors: Kumar Aditya , Thomas Stephanie , Wong Kirsten , Lo Sardo Valentina , Cheah Daniel , Tenerelli Kevin , Ferguson William , Topol Eric , Baldwin Kristin , Engler Adam
Published: July 2021

Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the non-coding 9p21 gene locus associated with increased risk of coronary artery disease (CAD) and myocardial infarction (MI). In addition, SNP correlations with sudden and arrhythmic death, even after accounting for patient and family history for CAD and MI, suggest an altered cardiac remodeling response. However, little is known about a possible cardiac phenotype as studies have largely focused on its effect on CAD and have trouble describing regulation with non-coding loci. Using induced pluripotent stem cell-derived CMs from patients that are homozygous risk/risk (R/R) and non-risk/non-risk (N/N) for 9p21 SNPs, we assessed cardiomyocyte (CM) function when cultured on hydrogels capable of mimicking the fibrotic stiffening associated with disease post-heart attack, i.e. stiffening from 10 kiloPascals (kPa) to 50 kPa. While all CMs independent of genotype beat synchronously on soft matrices, R/R CMs cultured on dynamically stiffened hydrogels exhibited asynchronous contractions versus N/N CMs in the same conditions. Dynamic stiffening reduced connexin 43 expression and gap junction assembly in R/R CMs but not N/N CMs. To eliminate patient-to-patient variability, we created an isogenic line by deleting the 9p21 locus from a R/R patient, i.e. R/R KO. R/R KO CMs maintained synchronous contractions and organized connexin 43 junctions after stiffening. The 9p21 locus suppresses the activity of the cell cycle regulator CDKN2A. p16, a protein produced by CDKN2A, prevents JNK phosphorylation (p-JNK), which in turn reduces gap junction expression in CMs and contributes to the development of arrhythmias in rabbit myocardium in response to stress. We observed that treatment with the p-JNK antagonist SP600125 after stiffening restored synchronous contractions and organized gap junction assembly to R/R CM. As a non-coding locus, 9p21 appears to repress connexin transcription, but only when the niche is stiffened as in disease. These data are the first to demonstrate that disease-specific niche remodeling can differentially affect CM function depending on SNPs within a non-coding locus.
Abstract 307: GJA1-20k Regulates Actin Cytoskeleton and Promotes Delivery of Connexin 43 to Cardiac Intercalated Discs
Authors: Basheer Wassim A , Xiao Shaohua , Zhang Shan-Shan , Epifantseva Irina , Fu Ying , Kléber André G , Hong TingTing , Shaw Robin M
Published: July 2021

Rationale:
Delivery of connexin 43 (Cx43) to the intercalated disc is a continuous and rapid process critical for intercellular coupling. By a pathway of targeted delivery involving microtubules and actin cytoskeleton rest stops, Cx43 hemichannels are efficiently trafficked to adherens junctions at intercalated discs. It has recently been shown that an internally translated isoform of Cx43, GJA1-20k, facilitates full-length Cx43 trafficking in cell lines, although the mechanism remains unknown.

Objective:
We explored the mechanism by which GJA1-20k regulates the trafficking of full-length Cx43 to intercalated discs.

Methods and Results:
In vivo overexpression of exogenous GJA1-20k, administered via AAV9-mediated gene delivery, increases the delivery of full length Cx43 to intercalated discs in mouse hearts. Using electron microscopy and fluorescence microscopy, together with biochemical co-immunoprecipitation, we found in micro-patterned HeLa cells and cardiomyocytes that GJA1-20k not only substantially increases the number and length of actin fibers, but can also rescue the effect of actin disruption. GJA1-20k complexes with actin and tubulin, improving microtubule targeting to cell-cell borders in the setting of actin disruption. Actin is also disrupted in acute ischemia-reperfusion (IR) injury. The ex vivo rescue potential of GJA1-20k was further tested in mouse hearts subjected to myocardial IR injury. As compared to control GFP and full length GAJ1-43k, only GJA1-20k gene transfer significantly improves the targeting of Cx43 to intercalated discs following IR injury.

Conclusions:
These results indicate that GJA1-20k positively modulates actin cytoskeleton to facilitate microtubule-based Cx43 trafficking machinery, promoting the delivery of full-length Cx43 to cardiac cell-cell junctions under normal and ischemic conditions. Therefore, up regulation of GJA1-20k is a potential therapeutic option to reverse the loss of Cx43 in IR injuries.
Abstract 325: Probenecid Downregulates Kidney Pendrin and AQP-2 and Potentiates Hydrochlorothiazide Diuresis
Authors: Soleimani Manoocher , Barone Sharon , Zahedi Kamyar , Xu Jie
Published: July 2021

Background:
Concurrent inactivation of the kidney Na-Cl co-transporter NCC and the Cl
-
/HCO
3
-
exchanger pendrin leads to significant salt wasting, whereas single deletion of NCC does not cause any significant salt wasting, indicating that pendrin mitigates the salt excretion caused by NCC inactivation. Probenecid is a uricosuric agent that, in addition, exhibits positive ionotropic effect in the heart and inhibits pendrin in the kidney.

Hypothesis:
Pretreatment with probenecid will inactivate/downregulate pendrin; therefore, leaving NCC as the main salt absorbing transporter in the distal nephron, and hence enhancing the hydrochlorothiazide (HCTZ) diuresis.

Results:
Male Sprague Dawley rats were treated with probenecid intraperitoneally (IP) at 250 or 100 mg/kg for 6 days and then received HCTZ while being maintained on probenecid for 4 more days. Urine output increased from 9.8 at baseline to 15.9 ml/24 hrs after 10 days of Probenecid at 250 mg/kg (p<0.01, n=5). Treatment with HCTZ alone for 4 days caused a mild diuresis, with urine output increasing to 13.8 ml/24 hrs (p>0.05, vs. baseline, n=5). However, rats pretreated with Probenecid for 6 days exhibited a profound diuresis when HCTZ was added for 4 additional days, with urine output increasing to 42.9 ml/day, a more than 300% increase vs. rats treated with either Probenecid or HCTZ (p<0.003 vs. both groups, n = 5). In the absence of pretreatment with Probenecid, the diuresis caused by concurrent Probencid plus HCTZ treatment was not different vs. HCTZ alone (p>0.05). Immunofluorescent, Northern and/or Western hybridization studies demonstrated a significant reduction in the expression of pendrin and AQP2 in kidneys of probenecid treated rats. At 100 mg/kg, Probenecid alone had no significant effect on urine output but caused a robust diuresis when HCTZ was added, with the urine output increasing from 9.93 baseline to 24.23 (p<0.001, n=7).

Conclusion:
Probenecid pretreatment downregulates pendrin and AQP2 and robustly enhances diuresis by HCTZ-mediated NCC inhibition in the kidney distal nephron. We propose that the combination of Probenecid and a thiazide derivative offers a powerful diuretic regimen for the treatment of fluid overloaded states such as CHF.
Abstract 348: Interleukin 13 is Required for Neonatal Heart Regeneration
Authors: O’Meara Caitlin C , Murphy Dana , Lemke Angela , Flister Michael J
Published: July 2021

Shortly after birth neonatal mice can fully regenerate their hearts, but this potential is lost in the first week of life. Cell cycle entry of existing cardiomyocytes is thought to be an essential mechanism enabling neonatal mouse heart regeneration. In previous studies we found that the cytokine interleukin 13 (IL13) was a an upstream regulator of differentially expressed gene networks during neonatal heart regeneration and stimulated cell cycle activity of cultured rat cardiomyocytes, suggesting that this factor might be important in neonatal heart regeneration
in vivo
. In the present study, we subjected wildtype and IL13 knockout mice to ventricular apical resection at one day of age and assessed heart regeneration 21 days post resection (dpr). Compared to wildtype controls, IL13 knockout mice failed to regenerate their hearts as determined by extensive scar formation at the ventricular apex. To gain insight into the mechanism of impaired regeneration, we quantified cardiomyocyte proliferation and expression of macrophage markers at 7 dpr. We found no difference in gene expression of macrophage markers in IL13 knockout mice compared to wildtype. Interestingly, IL13 knockout mice demonstrate a significant increase cardiomyocyte cell cycle activity as determined by phosphorylated Histone H3 (pH3) staining. This seemingly contradictory result appears to be due to an underlying developmental defect in IL13 knockout hearts. Cardiomyocytes in IL13 knockout mice appeared large and disorganized. Cardiomyocytes from IL13 knockout unoperated mice showed decreased pH3 staining and had increased expression marker of hypertrophic growth such as Nppb and Nppa. Histologically, hearts from IL13 knockout mice appeared to have a dilated cardiomyopathy phenotype. Collectively our data suggests that during heart development IL13 influences proliferative versus hypertrophic growth. We surmise that following neonatal apical resection in IL13 knockout mice the significant increase in cardiomyocyte proliferation is a compensatory attempt to repair the injury, but the underlying cardiomyocyte phenotype inhibits complete regeneration. These data are the first to report a role for IL13 in normal heart development and neonatal heart regeneration.
Abstract 357: Activation of Autophagic Flux Blunts Cardiac Ischemia/Reperfusion Injury
Authors: Cho Geoffrey W , Xie Min , Kong Yongli , Li Dan L , Luo Xiang L , Altamirano Francisco , Morales Cyndi , May Herman , Schiattarella Gabriele , Medina Jessica , Jiang Nan , Shelton John , Ferdous Anwarul , Gillette Thomas G , Hill Joseph A
Published: July 2021

Background:
Reperfusion injury accounts for a significant portion of myocardial damage in acute coronary syndromes. Autophagy, a process of cell catabolism, plays a vital role in the heart’s response to stress. We have reported that re-induction of ischemia/reperfusion (I/R)-suppressed cardiomyocyte autophagy with histone deacetylase (HDAC) inhibitors affords significant cardioprotection. However, as HDACs govern many processes and may have off-target effects, we set out to modulate autophagy in a manner independent of HDAC activity. Here, we hypothesized that induction of autophagy with a novel agent, Tat-Beclin, at the time of reperfusion, will reduce I/R injury and rescue cardiac function.

Methods:
Wild type and ATG7 (protein required for autophagic flux) knockout mice were randomized among 3 treatment groups prior to surgical I/R injury [45 min LAD artery ligation; 24h reperfusion]: vehicle control (VC), Tat-Scrambled (TS), or Tat-Beclin (TB). Each agent was delivered at coronary reperfusion. To define molecular mechanisms, cultured adult and neonatal rat ventricular cardiomyocytes (ARVMs/NRVMs) were subjected to simulated I/R.

Results:
Induction of cardiomyocyte autophagy at reperfusion reduced infarct size 20.1% (±6.3%, n=23, p<0.02 vs VC). This treatment was associated with improved systolic function (declines in fractional shortening: 19.8±3.7% VC; 18.7±2.1% TS; 8.5±1.7% TB, n=11, p<0.01 vs VC). In NRVMs subjected to I/R injury, cell death was reduced 41% (±6%, n=12, p<0.001 vs VC). Improvements correlated with increased autophagic flux measured by the marker LC3-II, particularly at the infarct border zone. Additional data suggested that autophagy rescues I/R injury through reduction of oxidative stress. ATG7 KO mice or NRVM depleted of ATG7 (RNAi) manifested significantly less cardioprotection.

Conclusion:
Direct induction of cardiomyocyte autophagy reduces infarct size and declines in contractile function. Autophagy rescues I/R injury in part through reduction of oxidative stress. Critically, this cardioprotection was observed when intervention occurred at the time of reperfusion, the clinically relevant context.
Abstract 38: Bioreactor Based Approach for Valve Tissue Engineering: Novel Application of Decellularized Porcine Pericardium
Authors: Amadeo Francesco , Polvani Gianluca , Agrifoglio Marco , Boschetti Federica , Pesce Maurizio , Santoro Rosaria
Published: July 2021

Background:
Aldehyde-fixed pericardium is commonly used in valve implant manufacturing. Despite its wide employment, this tissue undergoes chronic rejection that limits implant performance and durability. In this work, we employed a method to engineer a leaflet-like tissue by seeding aortic valve interstitial cells (VIC) into fixative-free decellularized pericardium using a bioreactor based approach.

Methods:
Following treatment with hypotonic buffer (Tris-HCl) to induce cell lysis, porcine pericardium was incubated with TritonX-100, to remove adipose tissue and then treated with sodium dodecylsulfate to wash cellular debris. DNA was removed by incubation into a DNAse I solution. Pericardium permeability was measured on samples before and after decellularization (pressure from 735 Pa to 2200 Pa). A direct perfusion bioreactor was employed to seed (3days, 3ml/min) porcine VICs (6.5E+5 cell/scaffold) into decellularized pericardium patches (6mm diameters) and perform long-term culture (up to 14 days, 0.03ml/min). Cell seeding efficiency (Day 3) and cell proliferation (Day 7-14) were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) staining, histological analyses and fluorescence staining for quiescent/activated VIC markers (DAPI, vimentin, α-smooth muscle actin).

Results:
Permeability tests revealed a significant increase in decellularized samples (1-way ANOVA
p
< 0.05), thus supporting its use in a direct perfusion bioreactor system. MTT staining revealed homogeneous cell seeding distribution, supported by DAPI staining, showing efficient cellularization through the whole patch volume. Computer-based cell nuclei counting revealed a significant cell increase from day 3 to 7 and 14 (
p
<0.05 1-way ANOVA). Immunofluorescence showed a marked reduction of αSMA in cells populating the inner layers.

Conclusions:
Our data show, for the first time, the capability to seed and culture VICs into a cell/fixative free pericardium with a direct perfusion system. Moreover, the use of pericardium treated with our decellularization procedure and recellularized under dynamic conditions supports a more physiological growth of VIC, as suggested by downregulation of αSMA in the inner pericardium layer.
Abstract 384: A Luminal Kinase Regulates Sarcoplasmic Reticulum Calcium Cycling
Authors: Pollak Adam , Wiley Sandra , Minamoto Shigeki , Heller Brown Joan , Dixon Jack
Published: July 2021

The mechanisms and machinery regulating calcium cycling through the sarcoplasmic reticulum (SR), a secretory pathway calcium storage organelle, remain incompletely understood, and further insight is critical for discerning normal and diseased heart function. An overlooked component is luminal SR protein phosphorylation. Our lab recently identified an atypical protein kinase in the secretory pathway lumen, Fam20C, and have demonstrated its role in calcium homeostasis for proper development of bone and teeth. This novel kinase phosphorylates Ser residues within highly conserved Ser-x-Glu/pSer (SxE) motifs, and is interestingly responsible for the overwhelming majority of secretory pathway protein phosphorylation. Therefore, we hypothesize that Fam20C phosphorylates proteins in the cardiac SR lumen, and that this phosphorylation will play an important role in SR calcium cycling. Indeed, we demonstrate that Fam20C phosphorylates SR proteins that have SxE sites, including Calsequestrin 2, Triadin, Sarcalumenin, Calreticulin, and Calumenin, all of which play important roles in SR calcium handling. Furthermore, we developed a cardiac specific Fam20C knockout mouse model. Following transverse aortic constriction (TAC) induced pressure overload, we find that Fam20C ablation reduces cardiac function and shows significantly increased signs of heart failure. Also, Fam20C ablation following TAC causes reduction in key regulators of SR calcium cycling, including SR Calcium-ATPase type 2a (SERCA2a) and phospholamban. Our results establish Fam20C as a novel cardioprotective signaling molecule, and open new avenues for potential therapeutic approaches to cardiovascular disease.
Abstract 395: A Synonymous Coding SNP Alters SCN5A Regulation by miR-24 and Associates With Non-Arrhythmic Death in Heart Failure
Authors: Zhang Xiaoming , Yoon Jin-Young , Morley Michael , Breheny Patrick , Bloom Heather , Dudley Samuel , Ellinor Patrick , Margulies Kenneth , London Barry , Boudreau Ryan L
Published: July 2021

Mutations disrupting
SCN5A
coding sequence cause inherited arrhythmias and cardiomyopathy, and SNPs linked to
SCN5A
splicing, localization and function associate with heart failure-related sudden cardiac death. However, the clinical relevance of SNPs that modulate
SCN5A
expression levels remains understudied. Recently, we generated a transcriptome-wide map of microRNA (miR) binding sites in human heart and evaluated their interface with polymorphisms. Among >500 common SNPs residing within miR target regions, we identified a synonymous SNP (rs1805126) adjacent to a miR-24 site within
SCN5A
coding sequence. This SNP is known to reproducibly associate with heart rhythm measurements, but is not considered to be “causal”. Here, we show that miR-24 potently suppresses
SCN5A
and that rs1805126 modulates this regulation. In further exploring the clinical significance of this, we found that rs1805126 minor allele homozygosity associates with decreased cardiac
SCN5A
expression and increased mortality in heart failure patients. Unexpectedly, this risk was not linked with arrhythmic sudden cardiac death, but rather, with clinical signs of worsening heart failure (e.g. reduced ejection fraction) and myocardial gene expression changes related to bioenergetics, inflammation and extracellular remodeling. Together, these data attribute a molecular mechanism to this firmly-established GWAS SNP and highlight a novel and surprising link between common variations in
SCN5A
expression and non-arrhythmic death in heart failure.
Abstract 413: Podoplanin Neutralization Improves Cardiac Remodeling and Function After Acute Myocardial Infarction
Authors: Cimini Maria , Garikipati Venkata N , Verma Suresh K , Benedict Cindy , Cheng Zhongjian , de Lucia Claudio , Wang Chunlin , Lucchese Anna Maria , Goukassian David , Kishore Raj
Published: July 2021

Variety of cardioprotective and reparative therapeutic approaches have emerged for the treatment of cardiac remodeling after myocardial infarction (MI). Here we propose a novel mechanism using a neutralizing antibody that target Podoplanin (PDPN), a platelet aggregation-inducing type I transmembrane glycoprotein, expressed on a cohort of myocardial cells that migrate to the infarcted area after MI and contribute significantly to scar formation. The PDPN+ cells were isolated from infarcted hearts two days after MI, using magnetic beads sorting. We tested in vitro the effect of PDPN neutralizing antibody (5μg/ml) in a transwell migration assay and the activation of monocytes co-cultured with PDPN+ cells. The neutralizing antibody decreased significantly PDPN+ cells migration. Monocytes co-cultured with PDPN+ cells produced high levels of IL1α and IL12, whereas treatment of co-cultures with podoplanin neutralizing antibody inhibited IL1α and IL12 production and increased IL9 and IL10 production, suggesting a switch form pro-inflammatory to anti-inlammatory phenotype. To tests the effect of podoplanin neutralizing antibody in vivo, C57BL/6 wild type mice were subjected to experimental MI and anti-PDPN antibody (25μg/ml) was injected i.p. on days 1, 2, 7 and 15 after MI and mice were scarified two months after. At 7 days after MI echocardiography revealed comparable ~30% of ejection fraction (EF) in control and antibody-injected mice. After one month EF% remained unchanged in control group and increased up to 45% in antibody-treated group, suggesting improvement in cardiac function. Histologically, in the control group the ischemic area was composed by fibrotic tissue highly positive for fibronectin and αSMA, whereas in the antibody-treated group revealed large number of survived, as well as proliferating myocytes expressing αSARC-actin and Phospho-H3. Further, there was a significant increase in CD31 positive cells in the infarct border-zone of antibody-treated vs. control hearts, suggesting increased angiogenesis. Our findings suggest that inhibition of PDPN during first two weeks after MI intensely enhances cardiac regeneration and angiogenesis. This may represent a new therapeutic support for the tissue renewal after MI.
Abstract 418: Genetic Rescue of Connexin 43 Remodeling in Muscular Dystrophy Cardiomyopathy
Authors: Himelman Eric , Contreras Jorge E , Shirokova Natalia , Fraidenraich Diego
Published: July 2021

Dilated cardiomyopathy has emerged as a major cause of lethality in Duchenne Muscular Dystrophy (DMD). Despite the growing influence on mortality, studies of dystrophic cardiomyopathy remain limited. We have found key evidence suggesting that aberrant expression of Connexin 43 (Cx43), a critical component of cardiomyocyte gap junctions, plays a detrimental role in DMD-cardiomyopathy. We showed Cx43 protein upregulation and lateralization away from the intercalated discs (IDs) to the lateral sarcolemma borders in both mouse (mdx) and human DMD cardiac tissues. Selective inhibition of the function of lateralized Cx43 protected Isoproterenol (Iso) challenged mdx mice from severe arrhythmias. We determined that Cx43 in mdx hearts is hypo-phosphorylated in a specific triplet of serine residues (S325/S328/S330). Reduction of phosphorylation in this same triplet promotes Cx43 lateralization and has been directly linked to cardiac ischemia, hypertrophy and arrhythmias. To investigate the role of the serine-triplet toward Cx43 remodeling and DMD cardiac pathology, we generated knock-in mutant mdx mice where the serine-triplet was modified to either non-phosphorylatable alanine (mdx:S3A) or phospho-mimetic glutamic acid residues (mdx:S3E). The changes in Cx43 phosphorylation status were confirmed by immunoblotting wherein mdx:S3E cardiac extracts prominently display slowly-migrating phosphorylated Cx43 bands while mdx:S3A extracts display a faster migrating isoform. We observed that 4 month-old mdx:S3E mice are protected from arrhythmias upon Iso challenge. Unlike mdx and mdx:S3A, mdx:S3E cardiomyocytes displayed strong Cx43 co-localization with N-Cadherin at the IDs, demonstrating improved Cx43 gap junction targeting. Together, these results suggest that the Cx43-serine triplet contributes toward Cx43 localization and cardiac dysfunction in mdx mice. This project will help identify possible molecular targets for pharmaceutical treatments to protect against cardiac failure and extend the lives of DMD patients.
Abstract 427: Alterations in the Mitochondrial Supercomplex in Pediatric Dilated Cardiomyopathy
Authors: Chatfield Kathryn C , Sparagna Genevieve C , Chau Sarah , Bennett Michael J , Chicco Adam J , Van Hove Johan L , Miyamoto Shelley D , Stauffer Brian L
Published: July 2021

Organization of the mitochondrial electron transport chain (ETC) into a protein “supercomplex” has been shown to be critical for optimal mitochondrial respiration, and is dependent on the phospholipid composition of the inner mitochondrial membrane. A close physical interaction between the ETC supercomplex and the fatty acid beta-oxidation system (FAO, which provides necessary reducing equivalents for ETC) has also been proposed. We have previously demonstrated that content of the primary mitochondrial phospholipid, cardiolipin, is altered in pediatric dilated cardiomyopathy (DCM), with evidence for its dysregulated biosynthesis. We hypothesized that altered cardiolipin content in pediatric DCM is correlated with altered supercomplex-associated ETC activity and mitochondrial fatty acid β-oxidation. A cross-sectional investigation was performed using myocardium from 16 children with DCM and 15 non-failing (NF) controls from the University of Colorado Heart Tissue Bank. Using blue native (BN) -PAGE with in-gel activity staining we demonstrated lower activity of supercomplex-associated complexes I (DCM 80% of NF, P<0.05) and IV (DCM 72% of NF, P<0.05) in pediatric DCM compared with NF controls. Using BN-PAGE and Western blot, as well as proteomic analysis of isolated supercomplex bands, we demonstrated interaction of the ETC supercomplex with FAO enzymes. Quantification of fatty acyl-CoAs was also performed in tissue from pediatric patients with DCM which demonstrated altered content of a subset of acyl-CoAs when compared to NF controls. We detected higher content of some C8, C10 and C12 CoAs in DCM compared with NF (P<0.05), with depletion of C18:1, C18:2, and C16 species (P<0.05). There was no difference between groups in free CoA or Acetyl-CoA. Taken together, these data suggest a potentially important interaction between the ETC supercomplex and long-chain β-oxidation enzymes, which may be altered on heart failure. We provide preliminary evidence for disrupted energy utilization in the failing pediatric heart.
Abstract 429: Assessment of Functional Mitochondrial Parameters in Single Cardiomyocytes Reveals Critical Events Not Identified in Cell Populations
Authors: Fajardo Giovanni , Kooiker Kristina , Zhao Mingming , Coronado Michael , Jung Gwanghyun , Hu Dong-Qing , Reddy Sushma , Bernstein Daniel
Published: July 2021

In cardiomyocytes (CMs), mitochondria play a dual role, maintaining the high energy supply required for rhythmic contraction, but also regulating critical cell death signaling. Impaired mitochondrial function can affect cellular homeostasis and contribute to sub-lethal injury; if mitochondrial impairment is more severe or persistent, pro-death pathways are activated. It is becoming increasingly clear, however, that within a population of cells there is considerable heterogeneity in mitochondrial function between individual CMs; and within a single cell there is also heterogeneity between individual mitochondria/mitochondrial regions. We have developed a high-throughput fluorescence imaging platform to quantitate single CM mitochondrial function in large numbers of cells, yielding information missed using standard assays that evaluate cell populations. When CMs are exposed to H
2
O
2
, they exhibit a dramatic hyperpolarization prior to the loss of mitochondrial membrane potential at the onset of cell contraction and death. There is marked heterogeneity in the timing of this response, with three distinct populations identified using Gaussian mixture models, and the duration of hyperpolarization longer for CMs that hypercontract earlier after H
2
O
2
exposure. This hyperpolarization is accompanied by a simultaneous increase in [Ca
2+
]
i
and is preceded by an increase in ROS. Standard methods, which average populations of cells, miss these responses. Blockade of MPT opening with cyclosporine A delays hypercontracture and cell death but does not prevent hyperpolarization. Finally, we have used our platform to track individual mitochondria/mitochondrial regions within a single live CM, identifying mitochondrial heterogeneity within a single cell, which is increased after exposure to H
2
O
2
, isoproterenol or in CMs from mice after ischemia-reperfusion injury. By tracking individual CMs and individual mitochondria within a single CM, we have opened a window to the complex heterogeneities in mitochondrial stress response.