bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 090: Long-term Therapy with a Bendavia (MTP-131), a Novel Mitochondria-Targeting Peptide, Normalizes Cardiolipin Biosynthesis and Remodeling in Left Ventricular Myocardium of Dogs with Advanced Heart Failure
Authors: Sabbah Hani N , He Quan , Gupta Ramesh C , Rastogi Sharad , Wang Mengjun , Zhang Keife
Published: July 2021

Background:
Cardiolipin (CL) is a signature phospholipid of the mitochondrial inner membrane and responsible for modulation of activities of various enzymes essential for oxidative phosphorylation. Cardiac CL is
biosynthesized
in a series of steps from phosphatidic acid and
remodeled
into a form which contains four 18:2 fatty acid chains, tetralinoleol CL [(18:2)
4
CL]. Long-term therapy with Bendavia (MTP-131), a novel mitochondria-targeting peptide, was previously shown to improve left ventricular (LV) function, increase ATP synthesis, and increase the activity and protein levels of mitochondria cytochrome c oxidase (complex IV) in LV myocardium of dogs with advanced heart failure (HF). This study examined the effects of Bendavia on total CL and (18:2)
4
CL in LV myocardium of dogs with HF.

Methods:
LV tissue was obtained from 12 dogs with microembolization-induced HF randomized to 3 months therapy with subcutaneous injections of Bendavia (0.5 mg/kg once daily, n=7) or saline (Control, n=7). LV tissue from 7 normal dogs was used for comparison. Total CL and (18:2)
4
CL were measured using electrospray ionization mass spectroscopy and quantified in nmol/mg of non-collagen protein (NCP).

Results:
Total CL was significantly decreased in LV myocardium of Control dogs compared to normal dogs (19.1±1.1 vs. 26.7±1.4 nmol/mg, p<0.05) as was (18:2)
4
CL (14.2±0.9 vs. 20.5±1.2 nmol/mg, p<0.05). Long-term therapy with Bendavia significantly increased both total CL (23.6±1.1 nmol/mg) and (18:2)
4
CL (17.8±1.0 nmol/mg) to near normal levels in LV myocardium of treated HF dogs compared to untreated HF Controls (p<0.05).

Conclusions:
Total CL and (18:2)
4
CL are decreased in LV myocardium of dogs with HF. Treatment with Bendavia normalizes total CL and (18:2)
4
CL. These findings are consistent with observed normalization of mitochondrial complex IV activity, normalization of rate of ATP synthesis and improvement of global LV performance in dogs with HF after long-term therapy with Bendavia.
Abstract 100: Delta Opioid Receptor Blockade Abolishes Exercise-Induced Cardioprotection Against Ischemia-Reperfusion Injury In Vivo
Authors: Borges Juliana P , Tristao Marina , Tibiriçá Eduardo V , Lessa Marcos A
Published: July 2021

Introduction:
Recent studies showed that exercise enhances myocardial tolerance to ischemia-reperfusion (I-R) injury via an opioid receptor-dependent mechanism. However, the specific subtype of opioid receptor involved in this response remains to be determined.

Methods:
Male Wistar rats were first divided into 2 groups: exercised and control. The exercised group underwent 4 consecutive days of treadmill training (60 min at 70% of maximal oxygen consumption). The exercised group was then divided into 4 subgroups: exercised (Exe; n = 10); exercised + kappa receptor antagonist (Exe + K; n=4); exercised + delta receptor antagonist (Exe + D; n=4); exercised + mu receptor antagonist (Exe + M; n=4). Control group was also divided into 2 groups: control (Ctr; n = 10) and control sham (Sham; n = 10). To induce I-R injury, anesthetized animals were submitted to a left thoracotomy and a 30 min interventricular coronary occlusion followed by 60 min of reperfusion. The hemodynamic parameters were recorded and infarct size was post-mortem determined by double staining using TTC/Evans blue and expressed as a percentage of the area at risk.

Results:
As shown in the figure, Sham group showed no infarct, Exe group showed a significant reduction in the infarcted area (27.6%) when compared to Ctr group (42.0%). The pretreatment with mu and kappa receptor antagonist did not alter the cardioprotective effect of exercise. However, the pretreatment with delta receptor antagonist prevented the exercise-induced cardioprotection.

Conclusions:
Endogenous opioid system is involved in cardioprotection conferred by acute exercise, and delta receptor subtype seems to play an important role in this response.
Abstract 101: The Continuous Administration of Sevoflurane Protects the Heart Against Ischemic Injury via Distinct Mitochondrial Mechanisms.
Authors: Lotz Christopher , Tischer-Zeitz Tobias , Frank Anja , Stumpner Jan , Roewer Norbert , Smul Thorsten M
Published: July 2021

Sevoflurane induced cardioprotection against ischemia/reperfusion (I/R) injury represents a well-established phenomenon. Salutary effects of the volatile anesthetic are not only limited to pre- or post-ischemic use, but clinical data also indicate its continuous administration as a beneficial mean. We investigated the hypothesis that the administration of sevoflurane during I/R-injury results in cardioprotection via the recruitment of distinct mitochondrial mechanisms. After IRB approval New Zealand White rabbits (9-12 weeks of age) received sevoflurane (1.0 MAC) or propofol (10 mg/kg/h) during 30 min of coronary artery occlusion followed by 3 h of reperfusion (n=7 per group). Cardiac mitochondria were isolated at the end of reperfusion. TTC-staining was used to determine the myocardial infarct size; in addition, mitochondrial O2 consumption was quantified and expressed as the respiratory control ratio (RCR). Mitochondrial electron transport chain (ETC) complex activities (I-IV) were assayed; including the analysis of an active to dormant (A/D) ratio of complex I. Statistics: ANOVA followed by posthoc Bonferroni, Mean ± SD. Our data indicate a significant infarct size reduction in animals receiving sevoflurane throughout the I/R-injury (p<0,05). The salutary effects were characterized by the maintenance of the RCR (p<0,05), as well as a preserved activity of complex I (p<0,05). Furthermore, the volatile anesthetic stabilized the A/D-ratio of the complex, indicating enhanced stability of the enzyme (p<0,05). Taken together, our findings support the notion that sevoflurane protects the heart against ischemic injury in a setting closely mimicking daily clinical work via the maintenance of mitochondrial respiratory physiology.
Abstract 102: The Detrimental Role Of iPLA2β In Ischemia/reperfusion Injury
Authors: Matsuga Hitomi , Yamaguchi Osamu , Tsujimoto Yoshihide , Otsu Kinya
Published: July 2021

Background:
Ca
2+
-independent phospholipase A2 (iPLA2) is crucial for a caspase-independent non-apoptotic cell death signaling pathway and involved in nuclear shrinkage induced by hypoxia. PLA2 family hydrolyzes the
sn-2
ester bond in phospholipids to release free fatty acids and lysophospholipids. PLA2 inhibitors are known to prevent ischemic-reperfusion (I/R) injury. Here, we attempted to clarify the role of iPLA2β in myocardial I/R injury using cardiac-specific iPLA2β-deficient mice.

Methods:
Pla2g6
gene-targeting vector was constructed using mouse BAC genomic library. The ES clones with targeted homologous recombination were injected into blastocyst mouse embryos. Floxed iPLA2β mice were crossed with transgenic mice expressing
Cre
recombinase under the control of the α-myosin heavy chain (
αMHC
) promoter to generate cardiac-specific iPLA2β-deficient mice (

pla2g6
flox/flox
αMHC-Cre
+

: CKO). We used

pla2g6
flox/flox
αMHC-Cre
-

littermates as controls (CTRL). We performed transthoracic M-mode echocardiographic analysis to examine the cardiac function of CKO and CTRL. Next, we subjected CKO and CTRL to I/R injury by using Langendorff-perfused mouse heart model. The heart was subjected to 30 minutes of global ischemia followed by 30 minutes of reperfusion. After the reperfusion, infarct area was determined with 1% triphenyltetrazolium chloride, and apoptotic cell death was detected by
in situ
TUNEL assay on heart sections. We exposed isolated adult cardiomyocytes from CKO and CTRL to various stimuli, such as Ca
2+
ionophore, H
2
O
2
and staurosporine, to examine the involvement of necrosis and apoptosis.

Results:
CKO and CTRL mice were born at the expected Mendelian ratio. There were no differences in cardiac function between CKO and CTRL. The functional recovery during reperfusion was significantly greater in CKO than in CTRL. CKO exhibited a significant decrease in the size of myocardial infarct. There were no significant differences in necrotic cell death induced by necrotic stimuli in vitro, while apoptotic cell death tended to decrease in CKO heart.

Conclusions:
These results suggest that iPLA2β exacerbates apoptotic cell death in I/R.
Abstract 106: Kcne2 Gene Deletion Predisposes To Sudden Cardiac Death
Authors: Hu Zhaoyang , Kant Ritu , Anand Marie , King Elizabeth , Krogh-Madsen Trine , Christini David , Abbott Geoffrey
Published: July 2021

Sudden cardiac death (SCD) accounts for an estimated 1000 deaths per day in the United States alone. SCD typically involves an ischemic substrate and culminates in a lethal ventricular arrhythmia.
KCNE2
potassium channel subunit gene variants are associated with cardiac arrhythmias including Long QT Syndrome, and impaired cardiac potassium channel function. KCNE2 is expressed in ventricular and atrial myocytes, but is also present in various epithelia, the potential cardiac implications of which have been largely overlooked. Here, we investigated whether
KCNE2
gene disruption could also perturb cardiac function via disruption of extracardiac roles. Targeted
Kcne2
deletion in mice caused hypercholesterolemia, glucose dysregulation and anemia - factors all known to predispose to myocardial ischemia in later life. In addition,
Kcne2
-/-
mice exhibited hyperkalemia, elevated serum angiotensin II, and prolonged T wave elevation - all potentially arrhythmogenic. Finally, in young female mice lacking overt baseline ischemia,
Kcne2
-/-
but not
Kcne2
+/+
mice exhibited polymorphic ventricular tachycardia, ventricular fibrillation and SCD within 20 minutes of reperfusion after brief coronary artery ligation. The findings demonstrate that
Kcne2
disruption creates a spectrum of abnormalities that produce both systemic and electrical substrates for SCD.
Abstract 118: Role Of Histone Trimethyl Demethylase Jmjd2A In Cardiac Hypertrophy And Heart Development
Authors: Liu Zhi-ping , Zhang Qing-Jun
Published: July 2021

Epigentic regulation via histone memethylation has been shown to play important role in embryonic development and pathogenesis of adult diseases. Methylation of histone H3 lysine 9 (H3K9) mediates heterochromatin formation and also participates in inhibition of gene transcription at euchromatic sites. Methylation of H3K9 is required for proper embryonic development as shown by various genetic studies of H3K9 methyltransferases in mice. Histone demethylases for methylated H3K9 have been shown to play important role in tumorigenesis. However, their roles in development and in adult cardiovascular diseases remain elusive. Jmjd2A is H3K9me3 specific demethylase. To study its biological functions, we generated mouse with conditional alleles of Jmjd2A. Our studies with post-natal heart specific Jmjd2A deletion and overexpression mouse lines indicated that Jmjd2A is required for pathological cardiac hypertrophy and the demethylase activity of Jmjd2A is required for its transcriptional activity. Jmjd2A also plays an important role during embryonic development. Mice with homozygous deletion of Jmjd2A exhibit partial embryonic lethality depending on the genetic background. In C57Bl6 (N>5) mouse strain, some of the Jmjd2A-null embryos die between embryonic day E10-E12 with heart failure phenotype. Deletion of Jmjd2A appears to have no effect on formation of linear heart tube and subsequent looping at E9.5. However, while WT or Jmjd2A het hearts continue to grow with appearance of ventricular and atrial septum and formation of trabeculae, Jmjd2A-null embryonic hearts have pericardial effusion, thin myocardium, lack of atrial and ventricular septation, and under-developed trabaculae. These failing hearts show reduced cardiomyocyte proliferation. Gene expression profiling suggests potential downstream effectors of Jmjd2A including cell cycle and “immediate early” genes. Our data demonstrate that Jmjd2A plays important roles during heart development and functions as a hypertrophic determinant in response to pathological stimuli in adult hearts.
Abstract 122: Increasing Short-term Cardiomyocyte Progenitor Cell Survival By Necrostatin-1 Does Not Further Preserve Cardiac Function
Authors: Feyen Dries , Gaetani Roberto , Liu Jia , Noort Willy , Martens Anton , den Ouden Krista , Doevendans Pieter , Sluijter Joost
Published: July 2021

Aim:
One of the main limitations for an effective cell therapy for the heart is the poor cell engraftment after implantation, which is partly due to a large percentage of cell death in the hostile myocardium. In the present study, we investigated the utilization of Necrostatin-1 (Nec-1) as a possible attenuator of cell death in Sca-1+ cardiac progenitor cells (CPCs).

Methods and Results:
Under oxidative stress conditions in vitro, Nec-1 pretreatment reduced necrotic cell death in Sca-1+ CPCs. In a mouse model of myocardial infarction, survival of CPCs 3 days after intra-myocardial injection was 39% higher (BLI signal, 71,665 ± 11,165 vs 117,138 ± 18,567 ph/s/cm2/sr) in cells pretreated with the Nec-1 compound. However, the increase in cell number did not translate into improved cardiac function at one month follow-up (EF %, 20.6±2.1 vs 21.4±2.5 for vehicle and Nec-1 treated CPC, respectively). We extensively investigated, but did not find, alternative effects to the pro-survival properties of the compound on the CPCs. Furthermore, CPCs rescued by Nec-1 remained functionally competent.

Conclusion:
A pharmacological pretreatment approach to solely enhance cell survival on the short-term does not seem to be effective strategy to improve cardiac cell therapy with Sca-1+ CPCs. Since the initial cell retention directly after injection is low (~10%), survival strategies could conceivably be more useful in a tissue engineering setting, in which they can exert their effects on larger number of cells.
Abstract 125: Genetic Deletion Of Telomerase Promotes Premature Cardiac Aging
Authors: Kim Junghyun , Sanada Fumihiro , Cimini Maria , Goihberg Polina , Hosoda Toru , Rota Marcello , Kajstura Jan , Anversa Piero , Leri Annarosa
Published: July 2021

Telomeropathies are a group of human diseases characterized by mutations in the telomerase gene, accelerated telomere attrition, and premature organ aging. Manifestations of telomere disease include bone marrow failure, liver cirrhosis, and lung fibrosis. It remains, however, to be documented whether loss of telomerase activity is coupled with alterations in cardiac structure and function. To address this issue, the heart of mice carrying a deletion of the RNA component of telomerase (Terc
-/-
mice) was studied at 3-13 months of age. This allowed us to define whether defects in telomerase function in cardiac stem cells (CSCs) and their progeny promote ventricular dysfunction independently from chronological age, which is typically associated with significant telomere erosion. For this purpose, the characteristics of the aging cardiomyopathy were defined first in 30-month-old wild-type mice (WT). Deteriorations in systolic and diastolic indices of myocardial contractility were detected in senescent mice by echo-Doppler, MRI, and invasive hemodynamics. Importantly, 6-month-old Terc
-/-
mice showed severe ventricular dysfunction comparable to that seen in 30 month-old WT. Telomere length in Terc
-/-
mouse CSCs was ~50% shorter than in age-matched WT cells but was comparable to that found in 30 month-old WT cells. The number of CSCs was 60% lower in Terc
-/-
than age-matched WT mice, and the fraction of BrdU-positive CSCs decreased 1.4-fold, from 25% to 14%. The absence of Terc led to a 50% reduction in myocyte turnover, which was coupled with myocyte hypertrophy and myocyte loss. BrdU labeling was reduced 60% in Terc
-/-
myocytes. Old CSCs formed a senescent progeny composed of cardiomyocytes, which carried markedly shortened telomeres and showed a severe depression in cell shortening and re-lengthening. Moreover, the renewal of endothelial cells was 75% lower in Terc
-/-
mice mimicking the rarefaction in capillary typically seen in the old myocardium. Our findings document that loss of telomerase activity is a critical determinant of cardiac aging with reduced cardiomyogenesis and vasculogenesis. These maladaptive responses may be operative in patients carrying mutations of telomerase.
Abstract 127: Mode of Injury Affects the Course of Regeneration and Repair in Neonatal and Adult Mouse Heart
Authors: Konfino Tal , Landa-Rouben Natalie , Leor Jonathan
Published: July 2021

PURPOSE:
Recent reports have demonstrated complete cardiac regeneration in newborn mice following resection of the cardiac apex. However, different types of injury could affect the mechanism of regeneration and repair.

HYPOTHESIS:
We aimed to test the hypothesis that the course of repair and regeneration after MI is different from apical resection in both neonatal and adult mouse heart.

Methods and Results:
Apical resection or permanent LAD coronary artery ligation was induced in 1-day-old or 12-week-old ICR mice. Echocardiography was used to confirm and monitor cardiac injury and remodeling. Mice were euthanized at different time points after operation, and hearts were harvested, processed, immunostained and compared with sham operated neonatal and adult hearts. Histological and immunohistochemical examination of both resected and infarcted neonatal hearts revealed inflammation and granulation tissue formation within 3 to 5 days. In the resected hearts, early regeneration was identified at the injured sites, and marked dedifferentiation of cardiomyocytes, represented by sarcomeric disassembly and marginalization, was evident around the injured areas. In addition, we noticed intensive proliferation of young cardiomyocytes which infiltrated the granulation tissue and formed a new myocardium within 21 days. In contrast, incomplete regeneration with residual small infract was detected 28 days after coronary occlusion. Echocardiography at 2,7,14 and 28 days after MI confirmed deteriorating LV function and LV remodeling with apical aneurysm formation. Surprisingly, 21 days after cardiac injury in adult mice, MI produced typical, thin, fibrotic scar whereas apical resection produced an apical tumor-like thick scar.

Conclusions:
The mode of injury, whether resection or infarction, affect regeneration and repair in both neonatal and adult mouse heart. Particularly, after apical resection, the newborn heart almost completely regenerates whereas regeneration is incomplete after MI, suggesting that infarction and subsequent inflammation might inhibit complete regeneration. Understanding these differences could be translated to development of new approaches to induce myocardial regeneration in adult heart.
Abstract 130: Secretion of Angiogenic and Anti-apoptotic Factors Accompanies Mesenchymal Stem Cell-mediated Enhancement of Contractile Function in Engineered Cardiac Tissues
Authors: Cashman Timothy , Mayourian Joshua , Costa Kevin D
Published: July 2021

Previous studies using three-dimensional engineered cardiac tissues (ECT) demonstrated beneficial effects of mesenchymal stem cells (MSC) on contractile function, recapitulating findings in animal studies and in clinical trials. But the mechanisms by which MSC effect this functional enhancement remain unclear. This study tested the hypothesis that MSC-mediated enhancement of ECT function involves paracrine signaling, independent of direct cell-cell interaction with cardiomyocytes (CM).

To create ECT, CM were isolated from neonatal rat ventricles, mixed with 2.0 mg/ml bovine type I collagen and 0.9 mg/ml Matrigel, and pipetted into a custom elastomeric mold with integrated force sensing end-posts. Two types of ECT were created: one containing 1.5 million CM (“CM-only”) and the other containing 1.5 million CM supplemented with 150,000 MSC (“MSC-CM hybrid”). Contractile function of CM-only ECT was then assessed during 2-Hz pacing either in the presence or absence of hybrid tissues sharing the same media. Media surrounding the tissues was also collected and analyzed via protein microarray (RayBiotech) combined with Ingenuity Pathway Analysis software.

CM-only ECT cultured for 5 days showed aligned and compacted structure with a cross-sectional area of 0.39 ± 0.03 mm
2
and a twitch force of 4.7 ± 0.7 N (mean ± SD; n = 6). In CM-only tissues co-cultured for 5 days in shared media alongside hybrid tissues, cross-sectional area was unchanged (0.42 ± 0.02 mm
2
, p = 0.17, n=3), but twitch force increased two-fold to 9.8 ± 6.5 N (p = 0.08). Threshold voltage for electrical pacing of CM-only ECT also decreased from 0.54 ± 0.12 V/mm to 0.39 ± 0.07 V/mm when co-cultured with hybrid ECT (p = 0.097). Protein microarray analysis of the shared co-culture conditioned media showed enrichment (relative to CM-only conditioned media) of the angiogenic factors VEGF (1240x) and IL-1 (8.6x) as well as the cardiac anti-apoptotic/pro-survival factors TNF (194x), bFGF (55x), and bNGF (14.9x).

In conclusion, these findings support paracrine signaling, independent of direct cell-cell contact, as one mechanism of MSC-mediated enhancement of CM function in rat ECT. Identification and isolation of defined cardiotropic factors may lead to novel molecular therapies for cardiac repair.
Abstract 131: C-kit+ Cells Generated From Failed Heart Do Not Improve Failed Heart Function
Authors: Zakharova Liudmila , Nural Hikmet , Nimlos James R , Popovic Snjezana , Feehery Lorraine , Gaballa Mohamed A
Published: July 2021

A pilot clinical study using autologous c-Kit+ cells showed improvement in cardiac functions in congestive heart failure (CHF), however, it is unclear if c-Kit+ cells isolated from CHF hearts are equally as potent as cells from controls. To test the potency of CHF c-Kit+ cells, myocardial infarction (MI) was created by permanent ligation of the left anterior descending coronary artery. Six weeks after MI, animals with left ventricle end-diastolic pressure (LVEDP) ≥20 mmHg and scar size ≥30% of left ventricle (LV) were designated as CHF rats. We found that CHF atrial explants generated less c-Kit+ cells compared to shams (15.7% vs. 11% sham vs. CHF). CHF c-Kit+ cells exhibited elevated levels of epicardial to mesenchymal transition markers, including Snail (2.5 fold) and Pai1 (3 fold), while the expression level of epithelial marker, E-cadherin was 3 fold lower in CHF c-Kit+ cells. Moreover, CHF c-Kit+ cells exhibited reduced gene expressions of pluripotency markers; 2.1 fold decrease in Nanog and 4.5 fold decrease in Sox 2 compared to sham cells. To evaluate the potency of the c-Kit+ cells, 1 x 10
6
cells isolated from CHFs or shams were delivered to 3 weeks post-MI CHF hearts. Cells were pre-labeled with GFP to enable their tracing in vivo and delivered to the infarcted myocardium via left coronary vein by a retrograde coronary sinus cell infusion (RCI). RCI delivery resulted in a cell distribution of LV (30%), right atrium (30%) and right ventricle (20%), while only 10% of cells were found in a left atrium. Three weeks after cells delivery, rats transplanted with sham c-Kit+ cells showed improved LVEDP (29.4 ± 6 vs. 11.7 ± 3.5 mmHg, CHF vs. CHF+ sham c-Kit+ cells) and a rise in peak rate of pressure (dPdt max) (3988 ± 520 vs. 5333 ± 597 mmHg/s). In contrast, no functional improvement was detected in rats transplanted with CHF c-Kit+ cells. Histological analysis demonstrated that transplanted c-Kit+/GFP+ cells were mostly incorporated into blood vessels and co-localized with endothelial marker vWf, and α-smooth muscle actin. Our results showed that left coronary vein is an efficient route for c-Kit+ cell delivery and that c-Kit+ cells isolated from CHF rats are less potent when transplanted in chronic heart failure rat model compared to those isolated from control.
Abstract 146: Glucagon-like Peptide-1 Receptor Activation Promotes Dual Benefits For Reversing Microvasculopathy And Mitochondrial Damage In Diabetic Heart.
Authors: Monji Akio , Bando Yasuko K , Mitsui Toko , Aoyama Morihiko , Kawase Hiroya , Murohara Toyoaki
Published: July 2021

PURPOSE:
Glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (Ex4) is a remedy for type 2 diabetes mellitus (T2DM). Ex4 ameliorates cardiac dysfunction in preclinical and clinical settings. However, it remains unclear whether the impact of Ex4 on cardiac remodeling in diabetic cardiomyopathy (DMC), of which primary characteristics are microvasculopathy and mitochondrial damage.

Methods and Results:
Diet-induced T2DM (DIO) mice and age- and gender-matched lean control mice were allocated into EX4 (24 nmole/kg/day for 40 days; DIO-Ex4 and LEAN-Ex4) and vehicle groups (DIO-veh and LEAN-veh). We first confirmed the GLP-1R expression in every single chamber of mouse heart by immunoblotting and PCR. Ex4 treatment ameliorated both systemic and cardiac insulin resistance without affecting body weight in DIO. Cardiac capillary density of DIO-veh was reduced compared to those LEAN-veh, which were reversed by Ex4 treatment. Tube formation assay and immunoblot analysis using culture endothelial cells revealed that Ex4 directly enhanced in vitro angiogenesis in a PKA/eNOS-dependent fashion. Systolic and diastolic left-ventricular (LV) dysfunctions observed in DIO-veh were restored by Ex4 with decline in LV wall thickening. Myocardial fibrosis detected using sirius-red staining and tissue oxidative stress detected by a fluorescence indicator DHE were attenuated in DIO-Ex4. Of note, analyses using transmission electron microscopy and a fluorescence indicator for damaged mitochondria (mitotracker red) revealed that Ex4 treatment reversed cardiac mitochondrial remodeling and increased healthy mitochondria. Ex4 treatment modulated cardiac oxidative stress balance by upregulating antioxidative molecules (SOD, thioredoxin, glutathione peroxidase) and reduction of NOX4 level; whereas it had no influence on NOX2 level.

Conclusions:
Ex4 enhances cardiac angiogenesis via GLP-1R-mediated activation of PKA/eNOS axis and accelerates reverses remodeling of myocardial mitochondria, at least in part, via its facilitating effects on antioxidative defense.
Abstract 164: Microdomain Specific Effects of Transient Receptor Potential Channels on Pathological Cardiac Hypertrophy and Myocyte Contractility
Authors: Makarewich Catherine A , Zhang Hongyu , Gao Hui , Correll Robert N , Duran Jason M , Molkentin Jeffery D , Houser Steven R
Published: July 2021

Hypothesis:
Ca2+ influx through transient receptor potential canonical (TRPC) channels and L-type Ca2+ channels (LTCCs) within caveolin-3 (Cav3) stabilized signaling microdomains provide a unique source of Ca2+ to activate pathologic cardiac hypertrophy through calcineurin (Cn)-mediated nuclear factor of activated T-cells (NFAT) signaling. We suggest that a distinct and separate population of TRPC channels localized in excitation-contraction (EC) coupling microdomains may have potent effects on myocyte contractility independent of Cav3 signaling domains.

Methods and Results:
Membrane localization studies and immunohistochemistry show that TRPC channels and LTCCs co-localize to Cav3 signaling microdomains. To explore a role for these caveolae based Ca2+ channels in the initiation of Cn-NFAT signaling we used an adenoviral NFAT-GFP reporter in cultured adult feline myocytes (AFMs). Infecting AFMs with ad-TRPC3 dramatically increased NFAT translocation, which was inhibited with dominant negative ad-dnTRPC6. Expression of a Cav3 targeted LTCC blocker (ad-Cav-Rem) reduced NFAT translocation while a targeted LTCC activator (ad-Cav-β2a) significantly increased NFAT activation. Neither LTCC modulator had significant effects on Ca2+ current or contractility in AFMs but we found that the expression of TRPC3 reduced myocyte contractility and induced spontaneous Ca2+ spark activity that was exacerbated by the DAG activator OAG. Moreover, dnTRPC6 blocked spontaneous Ca2+ sparks even in the presence of OAG. Immunohistochemistry analysis revealed the presence of TRPC channels in transverse tubules, consistent with the idea that they could have direct effects on EC coupling microdomains.

Conclusions:
Our data show that TRPC channels and LTCCs co-localize to Cav3 signaling domains where they generate a unique Ca2+ microenvironment that directly regulates Cn-NFAT signaling. Our findings also suggest that a separate and distinct population of TRPC channels within EC coupling microdomains cause reduced myocyte contractility by inducing SR Ca2+ leak and Ca2+ spark activity.
Abstract 184: Pressure Overload Activates Left Ventricular (LV) Intramyocardial Vascular Endothelial Cells and induces T Cell Infiltration into the LV
Authors: Alcaide Pilar , Grodecki-Pena Anna , Knapp Andrew , Kershaw Tanya , Aronovitz Mark , Kapur Navin , Blanton Robert , Karas Richard H
Published: July 2021

Left ventricular dysfunction and Heart Failure (HF) are associated with systemic inflammation with clinical data showing that HF patients have higher levels of circulating pro-inflammatory cytokines. Recruitment of circulating T cells to tissues across the vascular endothelium is a key event in the inflammatory response, but whether it plays a role in the heart in HF is unknown. We hypothesized that pressure overload induced HF activates cardiac endothelial cells resulting in T cell recruitment into the left ventricle (LV). Using transverse aortic constriction (TAC), quantitative flow cytometry, immunohistochemistry, qPCR and real time live cell videomicroscopy, we examined mRNA and protein expression levels of endothelial cell adhesion molecules and the presence of T cell infiltrates in the LV
in vivo
, and also studied the T cell interactions with primary mouse heart endothelial cells (MHEC) under flow conditions
in vitro
, comparing Sham and TAC operated mice (6-10/group) during the course of HF. 48h after TAC, in the pre-hypertrophic state, no differences were observed in the recruitment of T cells in the LV. Interestingly, two and four weeks after TAC, when mice developed LVH and LV dysfunction (Fractional Shortening 25±13%), E-Selectin, VCAM-1 and ICAM-1 mRNA levels were significantly upregulated in the LV as compared to Sham mice (2.3, 2.8 and 4 fold, respectively), with notable enhancement of endothelial ICAM-1 protein levels in the LV intramyocardial vessels, and T cells infiltrated in the LV in response to TAC (P≤0.05 TAC vs Sham). Furthermore, T cells isolated from mice 2 and 4 weeks after TAC adhered to MHEC under flow conditions in significantly higher numbers than T cells from Sham mice (P≤0.01 TAC vs Sham). Systemically, the frequency of three different T cell subsets in the peripheral lymphoid organs was increased in TAC vs Sham mice, indicating activation of the adaptive immune response to pressure overload. Taken together, our studies indicate that activation of the heart vascular endothelium occurs in response to pressure overload resulting in T cell recruitment into the LV. Further studies will be needed to determine in the extent to which T cell recruitment into the heart contributes to the pathogenesis of HF.
Abstract 185: Muscle Enriched A-Type Lamin Interacting Protein (MLIP) mediates growth and function of the adult heart
Authors: Cattin Marie-Elodie , Thorn Stephanie L , Wang Jessica , Rau Christoph , Mak Esther W , DaSilva Jean N , Lusis Jake , Burgon Patrick G
Published: July 2021

The newly discovered Muscle-enriched A-type lamin interacting protein (MLIP) is abundantly expressed in the heart. Its biological function remains elusive but it may be of high relevance for cardiac function as it has no paralogous homologue suggesting no functional redundancy. To elucidate MLIP’s function, we generated both hemizygous MLIP and homozygous MLIP null mice. At 8 weeks of age, they both develop dilated cardiomyopathy characterized by enlarged hearts (heart to body weights MLIP+/+ 5.62mg/g vs MLIP+/- 10.73mg/g & MLIP -/- 11.03mg/g) that are functionally dilated (LVDD : +/+ 2.89mm; +/- 3.93mm; -/- 4.11mm) with reduced function (LVFS : +/+ 47%; +/- 31%; -/- 29%). Histological analysis shows no overt abnormalities for both MLIP+/- and MLIP-/- hearts. Preliminary data indicate a preserved cardiomyocyte size in MLIP+/- and MLIP-/- mice, suggesting that the increase in heart size might be due to hyperplasia rather than cellular hypertrophy. Moreover, cardiac gene expression of 12 week-old MLIP+/- and MLIP-/- mice reveals an involvement in pathways associated with cell cycle control and growth. Specifically both gene expression and western analysis demonstrated that the mTORC1 pathway, that promotes protein synthesis and cell growth in response to mitogenic signal, was dysregulated in MLIP null mice. To further characterize these growth pathways the MLIP +/+ MLIP +/- and MLIP -/- mice (n = 3-5/group) were treated with 20 μg/g/day of isoproterenol through an abdominally implanted Alzet micropump for 3 weeks to induce heart failure. All mice went under echocardiographic analysis and were characterized by western, gene expression and histological analysis. Our preliminary data reveal that MLIP null mice are resistant to isoproterenol-induced heart failure as their hearts showed no increase in mass, unlike the significant (p<0.01) increase in heart mass observed in both the control mice (31%) or the MLIP hemizygous mice (19%). No cardiac remodeling was observed in either the MLIP hemizygous or null mice in response to the isoproterenol treatment. In conclusion, MLIP is a newly discovered protein that impacts key pathways involved in cardiac growth and function of an adult heart failure model.
Abstract 189: Natriuretic Peptide Receptor-A Regulates Angiotensin-II Mediated NF-κB Via Mkp-1 Dependent Pathway
Authors: Venkatachalam Gopi , Subramanian Umadevi , Arumugam Parthasarathy , Vellaichamy Elangovan
Published: July 2021

Atrial natriuretic peptide (ANP) exerts local anti-hypertrophic activity in heart tissue by binding to natriuretic peptide receptor (NPR)-A. However, patients with cardiac hypertrophy and congestive heart failure have elevated plasma and tissue levels of ANP and brain natriuretic peptide (BNP) along with Angiotensin II (Ang II). However, the rationale behind the impaired action of ANP in diseased state is not well understood. In this study, we sought to examine the signaling mechanism by which Ang II modulates local anti-hypertrophic effect through inhibition of
Npr1
gene, which codes for NPR-A, in the heart. Hence,
in vivo
, Wistar male rats (n=8/group) were administered suppressor dose of Ang II (50ng/kg/min) for 14 days through implanted mini-osmotic pumps. Also,
in vitro
, H9C2 (2-1) cardio myofibroblast cells were exposed to Ang II (10
-7
M) for 20 hours. Upon treatment with Ang II, the mRNA and protein expression of
Npr1
(p<0.01) was decreased with significant increase in expression of AT1R (p<0.01) in the heart tissues. In addition, a concomitant decrease in cGMP activity and production in isolated heart tissue membrane preparation was found in Ang II infused rats. Moreover, Ang II infusion causes a suppression of MKP-1 phosphatase; while enhancing the phosphorylation of ERK1/2 (p<0.01) and NF-κB (p<0.01) proteins. Similarly, H9C2 cells exhibited the hypertrophic growth with increased expression of AT1R and activation of ERK1/2 proteins on stimulation with Ang II. Furthermore, gene silencing using siRNA-NPR-A prior to Ang II treatment augmented the translocation of NF-κB and activation of ERK1/2 (3-fold). Whereas, pre-treatment with losartan or cGMP analog 8-Br-cGMP, an activator of cGMP-dependent protein kinases, abolished the stimulatory effects of Ang II on AT1R, NF-κB nuclear translocation and phosphorylation of MAPK, but activated the MKP-1 phosphatase. These results suggest that NPRA-cGMP signaling exerts inhibitory effects on Ang II by antagonizing the upstream signaling pathways and by activation of MKP-1 to counter-regulate NF-κB and MAPKs through cGMP dependent mechanism; thereby mediate local anti-hypertrophic activity in cardiac hypertrophy.
Abstract 191: Phosphodiesterase 9a Modulates Cardiomyocyte Natriuretic Peptide-Stimulated cGMP Pool and Cardiac Hypertrophy
Authors: Lee Dong I , Sasaki Takashi , Zhu Guangshuo , Rainer Peter R , Zhang Manling , Danner Thomas , Jo Su-huyn , Bedja Djahida , Takimoto Eiki , Kass David A
Published: July 2021

Cardiac hypertrophy in response to pathologic stress is a major contributor to heart disease. Many studies have revealed that compartmentalized cyclic guanosine monophosphate (cGMP) regulation by phosphodiesterases (PDEs) can potently modify acute and chronic cardiac stress responses. PDE5a normally regulates cGMP generated from nitric oxide (NO)-stimulated soluble guanylate cyclase (sGC) but not natriuretic peptide (NP)-stimulated cGMP. Its inhibition blunts maladaptive hypertrophy and remodeling. Regulators of NP-cGMP remain uncertain. Here we reveal that PDE9a, a highly cGMP-specific PDE (×100 affinity vs PDE5a) that is expressed predominantly in the brain modulates myocyte cGMP as well, targeting NP-stimulated pools. PDE9a gene expression is observed in rat neonatal cardiac myocytes (RNCM) and adult mouse myocytes. Protein expression was identified in myocytes by immunohistochemistry, using gene silencing models as a negative control. PDE9a expression is upregulated by various hypertrophic stimuli (phenylephrine (PE) or endothelin-1 (ET-1), and observed in myocytes from pressure-overloaded whole myocardium. PDE9a inhibition with a selective antagonist (PF-9613) or gene silencing significantly abrogated PE- or ET-1-dependent upregulation of pathological-hypertrophy fetal genes and NFAT activity in RNCM, and PDE9a inhibition attenuated ET-1-induced hypertrophy in adult mouse myocytes. PF-9613 had no impact in cells lacking PDE9a (from knockout mice) or after gene silencing. Biochemical and cGMP-sensitive fluorescent probe studies shows PDE9a inhibition augments ANP- but not NO-dependent cGMP stimuli in RNCM and adult myocytes. PDE9a knockout (KO) mice were protected against hypertrophy/remodeling after pressure-overload, accompanied by a rise in myocardial cGMP. In PDE9a KO mice also exhibited less fibrosis and reduced expression of fibrotsis-related genes over wild-type mice. Together, these data identify PDE9a as a novel regulator of myocyte cGMP and hypertrophy that impacts a different compartmentalized cGMP pool to that by PDE5a. Our observations may provide a novel therapeutic approach in the treatment of heart failure.
Abstract 193: Regulation of Mitochondrial KATP Channels In Pressure Overloaded Mouse Hearts
Authors: Shi Nian-Qing (Nan) , Gao Jianjiong , Ou Wen-Jie , Sabat Grzegorz , Xu Dong
Published: July 2021

Background:
ATP-sensitive potassium channels (KATP), existing in both sarcolemmal and mitochondrial inner membranes, are thought to serve as metabolic sensors of the cell. In pathological hypertrophy, the heart requires increased workload to pump blood to meet the body’s needs. However, roles of KATP in hypertrophy stress signaling are not fully understood.

Methods & Results:
We aimed at investigating regulation of SUR2, the regulatory subunit of KATP, via a systems biology approach. We hypothesized that mitochondrial KATP mediates the protective response in hypertrophied hearts. A pressure-overload model by transverse aortic constriction (TAC) was employed to induce hypertrophy in SUR2 knockout (SUR2KO) males. These mice had enlarged left ventricles at baseline and developed further hypertrophy post TAC. Hypertrophied KO hearts exhibited significantly higher mortality, cardiac dysfunction, reduced mitochondrial DNA content and elevated ROS formation, which were associated with the altered SUR2 (mitoSUR2) variant expression. Among the mitoSUR2 variants, a 55-kDa mitoSUR2A variant was significantly up-regulated 4-fold in both TAC and SHAM treated KO hearts. We then analyzed whether the mitoSUR2 variants were associated with a key ROS regulator ShcP66 variant, ShcP52, using quantitative BRET assays. Our data showed that ShcP52 could directly bind to the mitoSUR2 variants. Our data indicates that these mitoSUR2 variants are involved in mediating ROS signaling. Comparative proteomic profiling and subsequent canonical pathway enrichment analyses revealed that mitochondrial function was the top affected system among the 41 identified pathways in SUR2KO hearts. Semi-quantitative comparisons between KO and WT data showed that the ETC activity was severely disturbed in SUR2KO-TAC mitochondria. Further data mining identified compromised cardiac parameters, affected molecular functions and possible diseases in hypertrophied SUR2KO hearts.

Conclusions:
Our novel findings reveal that altered mitochondrial SUR2-based KATP activities likely contribute to the hypertrophic phenotype and abnormal mitochondrial function in SUR2KO hearts, suggesting that these channels play an essential role in mitochondrial remodeling and energy sensing.
Abstract 198: Stim/orai Channel Complex In Normal And Hypertrophied Adult Cardiac Myocytes
Authors: Hulot Jean-sebastien , Saliba Youakim , Fauconnier Jeremy , Keck Mathilde , Bénard Ludovic , Marchand Alexandre , Farès Nassim , Lompré Anne-Marie
Published: July 2021

Cardiac myocytes use Ca2+ not only in excitation-contraction coupling but also as a signaling molecule for cardiac growth. It is however unclear how Ca2+ triggers signaling in cardiac myocytes in the presence of the rapid and large Ca2+ fluctuations that occur during excitation-contraction coupling. We have recently reported that stromal interaction molecule 1 (STIM1), a calcium sensor in the sarcoplasmic reticulum, is a critical element in promoting cardiomyocyte growth through the control of a previously unrecognized sarcolemmal voltage-independent current. Our goal was to characterize the plasma membrane partners that contribute to STIM1-dependent currents in normal and hypertrophied adult cardiac myocytes.

We firstly characterized the expression profile of TRPCs and ORAIs proteins in both normal and hypertrophied ventricular myocytes from abdominal aortic banded rats. The expression of TRPC1, 3, 4, 5 and 6 and of ORAI1 - 3 was identified with an up-regulation of TRPC1 in hypertrophied cells. We then used a non-viral method to deliver cy3-tagged siRNAs to ventricular myocytes to knock-down the candidate channels. The cardiac myocytes were isolated and whole cell patch-clamp technique as well as Fura-2 imaging were performed to measure the currents in cy3-labelled and in control cardiac myocytes. Our data demonstrated that the drug-inducible STIM1-dependent store-operated calcium entry was marginal in normal myocytes but was enhanced in hypertrophied myocytes. In addition, a basal inwardly rectifying current was observed in hypertrophied but not in control myocytes. This spontaneous current was recorded in the absence of calcium store depletion, required STIM1 expression and meets the characteristics of Arachidonate-Regulated Calcium selective (ARC) channels. ORAI silencing completely inhibited both store-dependent and store-independent currents suggesting the critical implication of ORAIs whereas TRPCs were only marginally involved.

In conclusion, ORAI channels are involved in STIM1-dependent currents observed in hypertrophied adult cardiac myocytes. In addition to store-operated calcium entry, the STIM1/ORAI complex provides an alternative, store-independent pathway for agonist-activated Ca2+ entry.
Abstract 203: Sodium Hydrogen Exchanger Isoform Switching And Kchip2 Upregulation In Elderly Porcine Atria
Authors: Kant Ritu , Hu Zhaoyang , Malhotra Jaideep K , Krogh-Madsen Trine , Christini David J , Heerdt Paul M , Abbott Geoffrey W
Published: July 2021

Aging-associated changes in the heart contribute to a wide variety of cardiovascular diseases. The aging atrium becomes increasingly more susceptible to development of atrial fibrillation, and even its pharmacological response to therapeutic drugs alters over time. Changes in structure, function, and pharmacology of the heart can potentially be tied to specific changes in gene expression with aging. Here, seeking molecular correlates of myocardial aging processes, we performed global “whole transcript” analysis of 25,388 genes using 572,667 probes to compare the left atrium (LA) transcriptomes of young adult (9 months old) versus elderly (10 years old) female Sinclair swine. The two genes exhibiting the largest increase in LA expression with aging were NHE2 (9.2-fold; n = 3-4; P = 0.00001) and KCNIP2 (3.8-fold; n = 3-4; P = 0.0002). Real-time qPCR recapitulated the NHE2 results and revealed strongest upregulation with aging in LA, and age-independent expression in the ventricles. NHE2 encodes sodium hydrogen exchanger isoform 2, which was previously considered to not be expressed in mammalian heart, the favored cardiac isoform being NHE1. NHE1 is considered to be important for reperfusion injury after ischemia but, surprisingly, has been a relatively disappointing therapeutic target in clinical trials. Here, we found NHE1 transcript expression in all chambers, but no upregulation in aging heart, raising the possibility that NHE2 upregulation could negatively impact the pharmacological responsiveness of aging tissue to NHE1-specific inhibitors. KCNIP2 encodes potassium channel beta subunit KChIP2, which tightly regulates Ito density. Real-time qPCR indicated equal KCNIP2 upregulation with aging in the LA and RA, and aging-independent expression in the ventricles. In silico modeling predicted the increased KCNIP2 observed here would increase Ito sufficiently to shorten the atrial refractory period, an established substrate for atrial fibrillation. In sum, the findings uncover potential molecular substrates for aging-associated changes in cardiac function, and suggest possible novel therapeutic avenues for aging-related cardiovascular diseases.
Abstract 206: Intramyocardial Lipid Dysregulation in End Stage Heart Failure
Authors: Bedi Kenneth C , Lim Charles , Mesaros Clementina , Aziz Moez K , Blair Ian A , Margulies Kenneth B , Rame J. Eduardo
Published: July 2021

Background:
Advanced heart failure (HF) is characterized by metabolic abnormalities but studies conflict about the nature of these defects.

Methods:
We investigated the presence or absence of toxic intramyocardial lipid species using carefully procured human myocardium from lean, non-diabetic end-stage heart failure patients. Left ventricular myocardial samples were obtained at the time of heart transplant. Cases included idiopathic dilated (n=5) and ischemic (n=2) cardiomyopathy (n=7), and brain-dead organ donors without any history of heart failure (n=12). The failing hearts were examined before and after support with a left ventricular assist device (LVAD, n=7). Frozen samples were evaluated by liquid chromatography and high-resolution mass spectrometry (LC-HRMS) to identify lipid species and further quantitate their abundance. Samples for LC-HRMS were prepared using the Folch extraction method, and the samples were normalized to protein concentration via BCA protein assay. We selected for masses with p-values less than or equal to (p= .005) and a fold change greater than or equal to 2.5

Results:
Diacylglycerols (DAGs), ceramides, sphingosines, and cardiolipins (CLs) were lower in failing samples than in non failing. The figure below for L-Palmitoylcarnitine is representative of the quantitative changes in lipids observed between failing and non-failing groups.

Conclusion:
Excess intramycellular lipid accumulation is not present in advanced heart failure. A significant deficiency in cardiac lipid species has been identified in end stage non-diabetic heart failure. Future studies in diabetic and obese patients with advanced heart failure are warranted.