bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 78: Pnmt+ "Primer" Cells Give Rise to Cardiac Myocytes and Neurons in the Mammalian Heart: Development of New Experimental Tools for Cardiac Regenerative Medicine Applications
Authors: Varudkar Namita M , Xia Jixiang , Abukenda Ibrahim , Pfeifer Karl , Ebert Steven
Published: July 2021

Phenylethanolamine n-methyltransferase (Pnmt) catalyzes the conversion of norepinephrine to epinephrine, and thus serves as a marker for adrenergic cells. We employed a combination of immunofluorescent histochemical staining and genetic fate-mapping strategies to show that two separate Pnmt+ cell populations contribute to heart development. Intrinsic cardiac adrenergic (ICA) cells originate from the primary heart field, and contribute to pacemaking, conduction, and working (contractile) myocardium. A second population of cardiac Pnmt+ cells is derived from migrating neural crest. These neural crest adrenergic (NCA) cells appear to contribute to cardiac neurons. By adulthood, most of the Pnmt+ cells show a distinctively left-sided orientation in the heart, with nearly 90% of them being found in the left atrium and ventricle. Surprisingly large swaths of ventricular muscle are derived from Pnmt+ primer cells. Since this region of the heart is highly vulnerable to coronary artery disease and often sustains varying degrees of damage following myocardial infarction, we hypothesize that directed stem cell differentiation into Pnmt+ primer cells could serve as a valuable resource for repair and/or regeneration of left ventricular myocardium for heart disease patients. To test this hypothesis, we have generated stable recombinant mouse embryonic stem cell (mESC) lines that express various fluorescent marker proteins under the control of the endogenous Pnmt gene regulatory network. These cells can be rapidly expanded in culture, sorted, and used for transplantation studies in animal models to determine their therapeutic effectiveness. The cells can be induced along cardiogenic or neurogenic pathways in vitro, and the resulting Pnmt+ cells from each population can then be collected and tested in vivo. To achieve this goal, we have knocked-in a nuclear-localized enhanced green fluorescent protein into the Pnmt locus to create Pnmt-nEGFP recombinant mESCs and mice. We show that nEGFP expression is specifically expressed in Pnmt+ cells in vitro and in vivo. This strategy allows us to identify and isolate Pnmt+ cells to evaluate their effectiveness for cardiac regenerative medicine applications. .
Abstract 012: Directed Differentiation of c-kit+ Human Cardiac Stem Cells in vitro
Authors: Hong Kyung U , Al-Maqtari Tareq M , Moktar Afsoon , Vajravelu Bathri N , Bhatnagar Aruni , Bolli Roberto
Published: July 2021

Although transplantation of c-kit+ cardiac stem cells (CSCs) significantly alleviates post-myocardial infarction (MI) left ventricular dysfunction, the exogenous CSCs in the recipient heart do not generate cardiomyocytes robustly. Controlling the fate of transplanted CSCs and inducing their robust differentiation would be important in further improving the efficacy of cardiac stem cell therapies. We assessed the hypothesis that differentiation of human c-kit+ CSCs can be enhanced by programming them with cardiac transcription factors, including GATA4, MEF2C, Nkx2.5 and Tbx5. Transcription factors were introduced into CSCs in culture via lentivirus either individually or in combination. The transduced cells were then cultured for 1 or 2 weeks and analyzed for expression of 36 different markers of differentiated cardiac cell types by quantitative RT-PCR. Introduction of GATA4 led to marked increases in both early and late markers of cardiomyocyte differentiation, such as BNP and troponin T. By 2 weeks, GATA4 also significantly induced some of the smooth muscle cell markers, including calponin-1 and smooth muscle myosin heavy chain, as well as fibroblast markers. Interestingly, these gene expression changes induced by GATA4 were accompanied by dramatic morphological changes in CSCs. Overexpression of Tbx5 in CSCs resulted in induction of some of the cardiomyocyte markers specifically, although the number of genes differentially expressed was significantly less than those by GATA4. Interestingly, introduction of multiple transcription factors in combinations did not further increase the expression of cardiac marker genes. In conclusion, these findings suggest that cardiac gene programs can be activated in CSCs via introduction of defined transcription factors and that the pattern of expression of differentiated cardiac markers is unique to each transcription factor. However, none of the transcription factors alone was sufficient to induce differentiation of CSCs into mature cardiomyocyte-like cells, indicating that multiple and additional factors as well as temporal changes in their expression may be required for full differentiation and maturation of CSCs.
Abstract 022: Higher Baseline Progenitor Cell Counts in Familial Hypercholesterolemic Patients Compared to Healthy Controls
Authors: Ramjee Vimal , Bhatia Neal , Ghasemzadeh Nima , Guo Ying , Li Qunna , Vaughn Louette , Nell-Dybdahl Christine , Waller Edmund E , Brigham Kenneth , Wilson Peter W , Quyyumi Arshed , Le Ngoc-Anh , Sperling Laurence S
Published: July 2021

Background:
Endothelial injury from exposure to cardiovascular risk factors leads to atherosclerosis, however, an endogenous response to vascular injury characterized by circulating progenitor cells (CPCs) contributes to endothelial repair and regeneration. Although CPCs are known to be inversely related to cardiovascular risk, it is uncertain whether these risk factors are also a stimulus for CPC mobilization. We measured the number of CPCs in patients with familial hypercholesterolemia (FH) and compared them to patients with hyperlipidemia and healthy controls, with the hypothesis that CPC levels would be modulated by FH.

Methods:
Eight patients with FH on statin therapy (mean age, 48 ± 17 years) were compared with 16 age- and gender-matched hyperlipidemic patients on statin therapy, and 16 healthy volunteers. CPCs were analyzed using flow cytometry (BD FACS Canto II) for mononuclear cell (CD45dim) surface expression of CD34 and a combination of VEGF2R, CD133, and CXCR4 epitopes. Results are expressed as means ± SD or as proportions, and analysis of variance was used to detect differences between the three groups.

Results:
FH patients had higher circulating numbers of CD34+/CD133+ cells compared to hyperlipidemic (1.49 ± 1.01 vs. 0.88 ± 0.52 cells/µL, p = 0.04, respectively) and healthy counterparts (0.97 ± 0.61 cells/µL, p = 0.08). Similarly, FH subjects also had higher circulating levels of CD34+/CD133+/CXCR4+ cells compared to both the hyperlipidemic (0.68 ± 0.36 vs. 0.30 ± 0.22 cells/µL, p = 0.002, respectively) and healthy group (0.32 ± 0.25 cells/µL, p = 0.003).

Conclusions:
CPC counts, particularly of early CPCs characterized by CD133, and also those expressing the homing receptor, CXCR4, are higher in FH patients compared to age- and gender-matched healthy controls and patients with hyperlipidemia, independent of statins. This suggests an activation of reparative and regenerative reserve in FH subjects. The increased level of CPCs in FH subjects compared to hyperlipidemic patients may be a collective result of a higher level of lipid burden, lower therapeutic response to anti-hyperlipidemic pharmacotherapy, and chronic immune-activation due to ongoing subclinical endothelial injury in FH patients.
Abstract 025: Oxidative-stress Mediates Apoptosis in a Human Model of Danon Disease and Heart Failure
Authors: Perry Cynthia N , Han Sangyoon , Clegg Stacey D , Ouyang Kunfu , Deacon Dekker , Chen Ju , Gong Qiuming , Zhou Zhengfeng , Chi Neil C , Adler Eric D
Published: July 2021

Emerging evidence has highlighted the importance of autophagy, an intracellular recycling pathway, in regulating cardiomyocyte bioenergetics, function, and survival. However, the mechanisms responsible for cellular dysfunction and death in cardiomyocytes with impaired autophagic flux remain unclear. Danon disease is a familial cardiomyopathy associated with impaired autophagy due to mutations in the gene encoding lysosomal associated membrane protein type 2 (LAMP-2). To investigate the pathogenesis of Danon disease, we created iPS cells from a patient with a mutation in LAMP-2 who expired from heart failure. Danon iPS-derived cardiomyocytes recapitulated key features of the disease in vitro, including impaired autophagic flux, increased cell size, abnormal calcium handling, and increased expression of natriuretic peptides. Danon cells were noted to have significant amounts of mitochondrial oxidative stress and apoptosis, both of which could be abrogated by treatment with the antioxidant N-acetylcysteine. These results implicate mitochondrial oxidative stress and apoptosis in the pathogenesis of Danon disease. They also provide the basis for the use of antioxidants for Danon disease and other autophagy-related human diseases such as heart failure, diabetes, cancer, and neurodegenerative disorders.
Abstract 033: Cardio-Cerebro-Vascular Diseases: Prevention in Metabolic Syndrome Patients
Authors: Siegel Günter , Ermilov Eugeny
Published: July 2021

Background:
ALP and GGT are indispensable constituents of brain microvessel endothelial cells, expressed and induced by IL-6. High values are entangled in affecting blood-brain barrier functionality. Since SOD bound to brain basement membrane and matrix heparan sulfate proteoglycan, is implicated in β-amyloid efflux, we examined in a pilot study of 11 patients with metabolic syndrome (MS) whether these biomarkers can be beneficially influenced through an antioxidant phytochemical approach with
Ginkgo biloba
(EGb 761, 2 × 120 mg/d). Also, MS is associated with an increased risk of developing diabetes and cardio-cerebro-vascular diseases (CCVD) (Figure).

Methods:
Photometric methods, ELISAs, EIAs and laser ellipsometry were applied.

Results:
A 2-month medication led to brain/liver biomarker changes: ALP -14.8% (p < 0.004), GGT -11.3% (p < 0.010). Antioxidative biomarker changes: SOD +17.7% (p < 0.009), GPx +11.6% (p <0.001), oxLDL/LDL -21.0% (p < 0.002), 8-
iso
-PGF
2α
-39.8% (p < 0.003), MPO -29.6% (p < 0.014). Antiinflammatory biomarker changes: IL-6 -12.9% (p < 0.041), Lp(a) -26.3% (< 0.001), hs-CRP -39.3% (< 0.005), WBC -6.3% (< 0.023), MMP-9 -32.9% (< 0.042). Vasodilator biomarker changes: cAMP +43.5% (< 0.001), cGMP +32.9% (< 0.001), nanoplaque formation -14.3% (p < 0.008), nanoplaque size -23.4% (p < 0.0004).

Conclusion:
Since under ginkgo ALP and GGT were downregulated and SOD upregulated, and plasma biomarkers of oxidative stress, plaque stability and progression, inflammation, vasodilatory second messengers and lipid composition beneficially altered, this phytochemical may be estimated as complementary drug with potentially preventive character for CCVD.
Abstract 035: Mechanism of Chronic CaMKII Inhibition-Caused Regression of Heart Failure: Beneficial Effects on Neurohormonal Activation, Left Ventricular and Cardiomyocyte Contractile Function, [Ca
2
+]i Regulation and Beta-Adrenergic Modulation
Authors: Cheng Che Ping , Shao Qun , Cheng Heng-Jie , Callahan Michael F
Published: July 2021

Background:
Recent evidence indicates that Ca
2+
/calmodulin-dependent protein kinase II (CaMKII) is upregulated in heart failure (HF), contributing to electric, structural and functional remodeling. CaMKII has been proposed to be a therapeutic target for HF. However, the role and mechanism of chronic CaMKII inhibition (I) in HF is unclear. We assessed the hypothesis that CaMKII I improves cardiomyocyte function, [Ca
2+
]
i
regulation, and β-adrenergic reserve, thus limiting HF progression.

Methods:
We compared left ventricular (LV) and myocyte functional responses and plasma levels of norepinephrine (NE) over a period of 16 weeks (W) in 6 control (C) and 14 rats with HF induced by isoproterenol (ISO) (170 mg/kg sq for 2 days). After ISO for 12 weeks, HF animals were assigned to receive 4 W treatment with: placebo (saline) (n=6), KN-93 (70 µg/kg/day sq via mini pump) (n=6), or KN-92 (70 µg/kg/day sq via pump) (n=2), respectively.

Results:
Compared with C, ISO-treated rats had HF onset at 4 W after ISO and progressed to severe HF at 16 W with increased plasma NE (1398 vs 342 pg/ml), decreased ejection fraction (EF, 37% vs 62%) and LV contractility (E
ES
, 0.8 vs 1.3 mmHg/μl). LV time constant of relaxation (τ) (17.8 vs 10.6 ms) increased, accompanied with significant reductions in cell contraction (dL/dt
max
, 78 vs 151 μm/s), relaxation (dR/dt
max
, 59 vs 114 μm/s) and [Ca
2+
]
i
transient ([Ca
2+
]
iT
) (0.18 vs 0.28). HF myocyte response to β-AR stimulation (ISO, 10
-8
M) was attenuated with significantly less increases in dL/dt
max
(34% vs 79%) and [Ca
2+
]
iT
(19% vs 36%). The LV and myocyte dysfunction persisted in KN-92-treated HF group. In contrast, treatment with KN-93 significantly increased E
ES
(1.2 mmHg/μl) and EF (60%), decreased τ (12.1 ms) and corrected the elevation of plasma NE (319 pg/ml). Importantly, basal myocyte contraction (dL/dt
max
,147 μm/s), relaxation (dR/dt
max
,107 μm/s), and [Ca
2+
]
iT
(0.25) improved. ISO-induced increase in dL/dt
max
(71%) and [Ca
2+
]
iT
(32%) were augmented and close to normal control levels.

Conclusion:
Chronic CaMKII I prevents HF-induced sympathetic nervous system activation and improves LV and cardiomyocyte basal and β-AR stimulated contraction and relaxation, thus playing a salutary role at later stages of HF.
Abstract 043: Regulation of cardiac excitability by non-canonical Sonic Hedgehog signaling.
Authors: Cheng Lan , Covarrubias Manuel , Koch Walter , Riobo Natalia A
Published: July 2021

Introduction:
Induction of Sonic Hedgehog (Shh) in the myocardium post myocardial infarction promotes neovascularization and reduces infarct size. Shh signals through the 7-TM protein Smoothened (SMO), which we have found to couple to Gi proteins. Our goal is to investigate the effect of Shh on myocardial excitability and electrical remodeling and the role of SMO/Gi coupling.

Methods and Results:
We performed whole-cell patch clamp action potential and K+ currents measurements, biochemistry and volume-conducted ECG measurements in Langerdoff preparations using wild type and transgenic GiCT mice with inducible cardiomyocyte-specific impaired Gi protein signaling. In normal hearts and isolated cardiomyocytes, Shh reduced by 80% the isoproterenol-induced cAMP increase and protein kinase A (PKA) activation (p<0.01). Shh also induced a 2-2.5 fold prolongation of the action potential duration (APD) (p<0.001) and a reduction of the IK, slow (9.3±0.8 vs. 1.9±0.5 pA/pF; p<0.05). In Langerdorff-perfused hearts, Shh agonists induced a ~30% increase of the QT interval (p<0.01) and premature ventricular contractions (PVC) (0 out of 6 controls vs. 8 out of 8 Shh) and ventricular tachycardia (VT) (0 out of 6 controls vs. 7 out of 8 Shh), while pre-perfusion with KAAD-cyclopamine, a SMO inhibitor, reduced the arrhythmic episodes (PVC in 3 out of 8 and VT in 0 out 8). These effects we mediated by Gi proteins, as cAMP reduction, prolongation of the QT segment, and development of arrhythmias were abolished in hearts of GiCT mice.

Conclusion:
Our study demonstrates that acute Shh signaling in the heart leads to reshaping of the action potential (AP) and results in a dramatic increase of ventricular arrhythmias. Our data constitute the first evidence that acute, noncanonical Shh signaling may be implicated in the pathological electrical remodeling that sets the substrate for life-threatening arrhythmias in HF patients.
Abstract 053: Carbaprostacyclin Promotes Vascular Growth in Mouse Hindlimb Ischemia
Authors: Liu Qi , Terry Toya , Pan Su , Yang Zhongwei , Willerson James T , Dixon Richard A
Published: July 2021

Background:
Prostacyclin and its stable analogues are vascular protective agents that have been used clinically to reduce blood vessel constriction. Recent studies indicate they stimulate angiogenesis in response to tissue ischemia, but their role in arteriolar growth remains unclear. In the current study, we test the hypothesis that the prostacyclin stable analogue carbaprostacyclin (cPGI2) facilitates arteriolar growth in ischemic hindlimbs.

Methods and Results:
Catheters were attached to osmotic pumps in order to continuously deliver either cPGI2 or saline (control) locally into ischemic mouse hindlimbs for 7 and 14 days (n=5/group). At 7 days of cPGI2 delivery, live microscopic images showed more distinct structural remodeling at the arteriolar level in the cPGI2 group than in the saline group, including tortuosity of arteriolar-to-arteriolar connections and increased intersecting of adjacent arterioles. Vascular casting with a silicone radiopaque agent into the distal region of the infrarenal abdominal aorta revealed a more pronounced arterial anastomoses in the cPGI2 treated versus the saline treated ischemic legs. After immunofluorescence staining, laser scanning confocal microscopic analysis found more transmembrane proteoglycan NG2 positive vessels in cross sections of anterior thigh muscles in cPGI2-treated mice than in saline-treated mice. At 14 days, quantitative micro-computed tomography analysis indicated cPGI2-treated legs had markedly increased vascular volume (41.28 ± 2.22 vs 27.11 ± 2.85 mm3), significantly more blood vessels (0.16 ± 0.014 vs 0.09± 0.011 1/mm) and less distance between vessels (6.60 ± 0.52 vs 10.15 ±1.14 mm) than did saline-treated legs. To further verify the pro-arteriogenic effect of cPGI2, a quantitative histogram was generated and exhibited that cPGI2-treated ischemic legs have a significant increase in small vessels, with vessel diameter bins ranging from 40-60 microns. We similarly evaluated contralateral nonischemic legs and found no significant differences in vessel distribution in the cPGI2 and saline groups.

Conclusion:
Multi-imaging technical analyses indicated that local delivery of carbaprostacylin promotes arteriolar growth in mouse hindlimb ischemia.
Abstract 086: Estrogen Replacement Downregulates Cardiac Angiotensin II in an ACE-independent Manner in Oophorectomized mRen2.Lewis Rats
Authors: Wang Hao , Zhao Zhuo , Jessup Jewell , Lin Marina , MacNamara Lindsay , Ahmad Sarfaraz , Ferrario Carlos , Groban Leanne
Published: July 2021

The cardioprotective effects of estrogen are well recognized, but the mechanisms remain poorly understood. Accumulating evidence suggests that the local cardiac renin-angiotensin system (RAS) is involved in the development and progression of left ventricle (LV) remodeling and diastolic dysfunction (LVDD). Estrogen attenuates the effects of an activated circulating RAS, however, its role in regulating the cardiac RAS is unclear. We hypothesized that estrogen replacement limits the development of the LVDD phenotype after ovarian hormone loss by shifting the RAS from the pro-fibrotic angiotensin II (Ang II)/ Ang II type 1 receptor (AT1R)/converting enzyme (ACE) and aldosterone pathway to the anti-fibrotic, Ang-(1-7)/Mas/ACE2 pathway. Bilateral oophorectomy (OVX; n=17) or sham-operation (Sham; n=13) was performed in 4-week-old, female mRen2.Lewis rats. At 11 weeks of age, after the development of exacerbated hypertension, the rats were randomized and received either 17 β-estradiol (E2, 36 μg/pellet, 60 day release, n=8) or vehicle (OVX-V, n=9) for 4 weeks. E2 treatment attenuated the adverse effects of estrogen loss on tissue Doppler-derived diastolic indices, e′ and E/e′, without changing blood pressure. E2 treatment minimized the rise in circulating Ang II and aldosterone, following OVX. Chronic E2 also attenuated OVX-associated increases in cardiac Ang II, Ang-(1-7), and chymase expression, and in mast cell number as determined by real-time PCR, western blot, and immunohistochemical analyses. Neither OVX nor OVX+E2 altered cardiac expression of AT1R, or gene expression and activity of cardiac ACE. E2 treatment in OVX rats significantly decreased gene expression of MMP-9, ACE2, and Ang-(1-7) mas receptor, in comparison to sham-operated, intact and OVX littermates. In conclusion, E2 treatment inhibits upsurges in cardiac Ang II expression in the OVX-mRen2 rat, possibly in a non-ACE-dependent manner. Further studies are warranted to determine whether an E2-mediated reduction in cardiac chymase directly contributes to this response in OVX rats.
Abstract 092: Transgenic Expression of Heat Shock Protein 60 Results in Neonatal Death and Adult Heart Failure
Authors: Liu Shan-Wen , Chen Tsung-Hsien , Chen Yen-Ling , Chen Mei-Ru , Hsu Ching-Han , Lin Kurt M
Published: July 2021

Heat shock proteins (HSPs) are known for their enhanced expression and pro-survival role in cells under noxious stress. Only recently was it discovered that aberrant expression of HSPs was linked to the onset of cardiovascular diseases. HSP60, originally identified as one of the mitochondrial chaperonins, was shown involved in autoimmune, metabolic, and cardiovascular diseases, but the roles of HSP60 remain unclear in part due to the lack of HSP60 transgenic mouse models. This study aims to investigate the outcome of expressing HSP60 in cardiac tissues to elucidate the role of HSP60 in initiating apoptotic cardiomyopathy.

By using a conditional HSP60 transgenic mouse line (G-Lox-HSP60) and a Cre mouse line, human HSP60 expression in most mouse tissues, driven by the ubiquitous CAG promoter, was achieved. The ubiquitous expression of HSP60 was found to cause neonatal death during postnatal day one. In HSP60 expressing newborn mice, hemorrhagic cardiac myopathy was found, and some of these mice exhibited atrium septal defects. A greatly increased rate of apoptosis was found in cardiac tissues with HSP60 overexpression, suggesting a pro-apoptotic role for HSP60. A tamoxifen-inducible Cre mouse (Myh6-Cre/ESR) was used to induce HSP60 expression in the adult mouse heart, and the results showed that dilated heart failure developed within 8 weeks after the induction of HSP60 expression. By employing an isolated perfusion model, the basal contractility was found to be significantly enhanced by HSP60, and a cardiac protection against simulated ischemia and reperfusion injury was found in mice with the HSP60 induction for 4 weeks. Both increased contractility and cardiac protection were lost in mice 6 to 8 weeks after HSP60 induction.

Our results showed that HSP60 expression led to heart failure in neonatal mice and likely involved developmental defects and induced apoptosis. While expressing HSP60 in adult cardiac tissues only temporally improved cardiac function and exerted cardiac stress protection, hypertrophy and heart failure eventually occured in these mice. The G-Lox-HSP60 model that achieves expression of HSP60 in an inducible and tissue-specific manner will be useful in studies to investigate the roles and molecular details of HSP60 in various diseases.
Abstract 099: Exercise-Induced Cardioprotection Against Ischemia-Reperfusion Injury Is Abolished by Opioid System Blockage Using an In Vitro Rat Model
Authors: Borges Juliana P , Paes Lorena S , Tibiriçá Eduardo V , Lessa Marcos A
Published: July 2021

Introduction:
A single exercise session releases opioid peptides considered to be associated to cardioprotection. However, the link between exercise-induced cardioprotection and the opioid system remains unclear. Therefore, the study aims to investigate the involvement of opioid receptors in exercise-induced cardioprotection in rats submitted to myocardial injury by ischemia/reperfusion (I/R)
in vitro
.

Methods:
Male Wistar rats (250-300g) were randomly divided into 3 groups submitted to I/R: control (CTR; n=5), exercised (EX; n=5) and exercised treated by naloxone, a non-selective opioid receptor antagonist (EX+NAL; n=5). The exercised groups underwent 4 consecutive days of treadmill training (60 min at 70% of maximal oxygen consumption). After the last exercise session (24h), the hearts were removed and immediately installed on a modified Langendorff apparatus. A latex balloon was inserted in the left ventricle (LV) to monitor the LV developed pressure (%LVDP). All hearts underwent a period of 30 min of sustained global ischemia followed by 1 h of reperfusion. After reperfusion, LV was sliced and incubated 20 min in triphenyltetrazolium to measure infarct size.

Results:
Anova two-way showed that CTR group presented a larger infarct area when compared to EXE group (32.9±9.2%
vs
11.3±4.0% respectively; p<0.05). Naloxone treatment completely blocked the exercise-induced cardioprotection (28.1±8.0%). In respect to %LVDP, CTR and EXE+NAL showed significantly lower results compared to EXE group from 25 min of reperfusion.

Conclusions:
Our results indicate that opioid system is involved, at least in part, in cardioprotective effects of aerobic exercise in rats.
Abstract 113: Regulation Of Aortic Vasodilation By Sigma-1 Receptor Stimulation In Ovariectomized And Pressure Overloaded Rats.
Authors: Tagashira Hideaki , Matsumoto Takayuki , Taguchi Kumiko , Kobayashi Tsuneo , Fukunaga Kohji
Published: July 2021

Objective:
We previously reported that sigma-1 receptor (

σ
1
R

) expression in the thoracic aorta decreased after pressure overload (PO) induced by abdominal aortic banding in ovariectomized (OVX) rats. Here, we asked whether stimulation of

σ
1
R with the selective agonist SA4503 elicits functional recovery of aortic vasodilation and constriction following vascular injury

in OVX rats with PO.

Methods:
SA4503 (0.3-1.0 mg kg
-1
) and NE-100 (an

σ
1
R

antagonist, 1.0 mg kg
-1
) were administered orally for 4 weeks (once daily) to OVX-PO rats, starting from the onset of aortic banding. Vascular functions of isolated descending aorta were measured following phenylephrine (PE)- or endothelin-1 (ET-1)-induced vasoconstriction and acetylcholine (ACh)- or clonidine-induced vasodilation.

Results:

σ
1
R

expression in aortic smooth muscle and endothelial cells decreased significantly 4 weeks after PO in OVX rats (vs. Sham or OVX only group). SA4503 administration rescued PO-induced

σ
1
R decreases in the

descending aorta. SA4503 treatment also rescued PO-induced impairments in ACh- and clonidine-induced vasodilation without affecting PE- and ET-1-induced vasoconstriction. Ameliorated ACh- and clonidine-induced vasodilation was closely associated with increased Akt activity and in turn endothelial nitric oxide synthase (eNOS) phosphorylation. SA4503-mediated improvement of vasodilation was blocked by NE-100 treatment.

Conclusions:

σ
1
R

is downregulated following PO-induced endothelial injury in OVX rats. The selective

σ
1
R

agonist SA4503 rescues impaired endothelium-dependent vasodilation in the aorta from OVX-PO rats through

σ
1
R

stimulation, enhancing eNOS-cGMP signaling in vascular endothelial cells. These observations encourage development of novel therapeutics targeting

σ
1
R

to prevent vascular endothelial injury in postmenopausal woman.
Abstract 135: Pim-1 Modification Reverses Senescent Phenotype Of Hcpcs From Patients With Heart Disease
Authors: Mohsin Sadia , Khan Mohsin , Alkatib Monique , Nguyen Jonathan , Wallach Kathleen , Sussman Mark A
Published: July 2021

Rationale:
Adoptive transfer of human cardiac progenitor cells (hCPCs) can repair damaged myocardium and improve function in a pathologically challenged heart. hCPC’s isolated from multiple patients exhibit differences in growth kinetics, expression of cell cycle regulators and telomere length suggesting hCPCs with slow growth kinetics may compromise effective regeneration of damaged or aged myocardium. There is a dire need to reverse the senescent phenotype of hCPCs exhibiting slow growth kinetics to improve the outcome of regenerative therapy.

Objective:
Demonstrate that Pim-1 engineering reverses the senescent phenotype of human cardiac progenitor cells (hCPCs) with slow growth kinetics by increasing their telomere length and decreasing population doubling time and cell cycle inhibitors.

Methods and Results:
hCPCs positive for stem cell marker c-kit were isolated from heart biopsy samples from patients undergoing Left Ventricular Assist Device (LVAD) implantation. hCPCs isolated from multiple patients were characterized for growth kinetics, telomere length, expression of cell cycling regulators. hCPCs with slow growth kinetics exhibit decreased telomere length concomitant with increased population doubling time and upregulation of senescent markers compared to hCPCs with fast growth kinetics. hCPCs were engineered to express Pim-1-GFP (hCPCeP), a fused GFP version of the kinase by using a lentivirus expression system. Cell cycling measured by population doubling time was decreased in hCPCeP. Similarly, telomere lengths in hCPCeP were significantly increased relative to control hCPCs expressing GFP (hCPCe). Similarly, the expression cell cycling inhibitors including p16 and p53 is down regulated indicative of reversal of senescent characteristics.

Conclusion:
Ex vivo gene delivery of Pim-1 in hCPCS enhances telomere length and decreases population-doubling time and senescent phenotype. These changes are more significant and distinct in hCPCs with slow growth kinetics. Pim-1 modification of hCPCs with slow growth kinetics from patients with heart failure can augments their ability to regenerate damaged myocardium and address current limitations associated with senescent phenotype of aged hCPCs.
Abstract 147: Glucagon-like Peptide-1 Receptor Activation Reverses Cardiac Mitochondrial Remodeling In Advanced Type 2 Diabetes Via Suppression Of Nox4.
Authors: Monji Akio , Bando Yasuko K , Mitsui Toko , Aoyama Morihiko , Kawase Haruya , Murohara Toyoaki
Published: July 2021

PURPOSE:
Damaged cardiac mitochondria are one of the critical features of diabetic cardiomyopathy. We indeed demonstrated that diet-induced type 2 diabetes mellitus (T2DM) exhibited cardiac mitochondria remodeling, which were reversed by glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (Ex4) (submitted as another abstract to BCVS 2013); however, it remains unclear whether the mitochondrial remodeling induced by diabetes may be reversible even at an advanced disease stage and Ex4 may be strong enough to exhibit the cardioprotective effects on such a progressive T2DM.

Methods and Results:
Genetic T2DM model (KKAy mice of which casual blood glucose levels is more than 30 mmol/L) were allocated into exendin-4 (Ex4, 24 nmole/kg/day for 40 days; KK-Ex4) and vehicle groups (KK-v). In KK-v, their systolic left-ventricular (LV) function was normal range, whereas they exhibited diastolic LV dysfunction and myocardial hypertrophy, which were ameliorated by Ex4. Myocardial fibrosis and cardiac steatosis (detected by Oil-red-O-staining and anti-ceramide antibody) were reduced in KK-ex4. Transmission electron microscopy revealed that Ex-4 normalized cardiac mitochondrial remodeling in terms of morphology, size, and number observed in KK-v. Ex4 attenuated mitochondrial damage which was detected by its specific dye mitotracker-red and alternatively monitored by mitofusin (Mfn)1/Mfn2 ratio. DHE staining revealed that Ex-4 reduced cardiac oxidative stress observed in KK-v. Ex4 modulated cardiac oxidative stress balance by reduction of NOX4 level; whereas it had no influence on NOX2 level and antioxidative molecules (SOD, thioredoxin, glutathione peroxidase), contrarily to those cases observed in diet-induced T2DM heart.

Conclusions:
Mitochondrial remodeling induced by T2DM was found to be reversible even at an advanced disease stage and Ex4 was strong enough to exhibit the cardioprotective effects on such a progressive T2DM by attenuating cardiac oxidative stress, at least in part, by suppression of NOX4.
Abstract 151: A Novel Role for DDiT4L in Regulation of mTOR and Autophagy in the Heart
Authors: Simonson Bridget , Franchino Hannabeth , Knight Ashley , Rosenzweig Anthony , Das Saumya
Published: July 2021

Introduction:
DDiT4L is a known negative regulator of mTOR signaling in skeletal muscle; however its role in the heart is unknown. We have recently showed increased DDiT4L mRNA in a murine transgenic model of pathological but not physiological hypertrophy. Here we test the hypothesis that DDiT4L is a regulator of mTOR signaling in the heart and may play a role in pathological hypertrophy and heart failure.

Methods:
We investigated the regulation of DDiT4L in murine models of hypertrophy and in cultured neonatal rat ventricular cardiomyocytes (NRVMs). Loss and gain of function of DDiT4L in mTOR regulation and autophagy was investigated using confocal imaging, immunoblotting, and qRT-PCR in NRVMs.

Results:
DDiT4L gene and protein expression was increased four-fold in pressure overload hypertrophy (n = 4-6, p<0.001), but not in a swim model of physiological hypertrophy. DDiT4L gene expression also significantly increased in a genetic model of dilated cardiomyopathy model (n = 4, p<0.001). In NRVMs, DDiT4L was induced by cardiac stressors such as pathological stretch, hypoxia, and glucose deprivation (n = 3-5 in duplicate, p<0.05-0.01). Increased DDiT4L expression correlated with inhibition of mTOR signaling, and an increase in autophagy markers. siRNA ablation of DDiT4L revealed that inhibition of mTOR signaling by DDiT4L was necessary for glucose deprivation induced autophagy, as determined by imaging of GFP-LC3 autophagosomes (n = 3 in duplicate, p<0.01), and immunoblotting of autophagy markers. Conversely, adenoviral-driven overexpression of DDiT4L inhibited mTOR signaling and significantly increased basal autophagy (n = 3 in duplicate, p<0.05). In TAC mice, the increase in DDiT4L protein expression correlated to inhibition of mTOR signaling, increases in autophagy markers (p<0.01), and preceded the transition to LV dilation and HF.

Conclusion:
Our data suggests that DDiT4L expression is altered in diverse models of pathological hypertrophy and precedes the development of LV dilatation and overt heart failure. DDiT4L inhibition of mTOR and modulation of autophagy may play a role in the progression to heart failure. DDiT4L may represent a novel therapeutic target to prevent this transition.
Abstract 155: Mutations In ABCA8 Result In HDL Deficiency And Cholesterol Efflux Defects
Authors: Singaraja Roshni , van Capelleveen Julian , Hovingh Kees , Zhang Linhua , Dallinga-Thie Geesje , Tietjen Ian , Wong Kenny , Kastelein John J , Hayden Michael R
Published: July 2021

The molecular basis of extreme HDL-C levels is incompletely understood. To identify mutations in novel putative HDL genes, we sequenced select genes in 80 unrelated individuals with extreme low HDLc (HDL %ile≤10th) and as controls, 120 individuals with high HDL (HDL %ile≥90th). We identified two variants in ABCA8 in subjects with low HDL-C levels. The E18-2T>C mutation is located in a highly conserved region and gives rise to a disruption of a splice site. The G>C variant occurs at a site conserved in vertebrates, at position 64425884 (build HG18), and results in Pro609Arg substitution. No ABCA8 mutations were identified in HHDL individuals. These represent the first mutations identified in ABCA8, which belongs to the family of ATP binding cassette transporters. Family expansion followed by genotyping identified additional mutation carriers and first degree relative controls in whom segregation analyses were performed. Compared with controls, mutation carriers showed a significant 32.6% decrease in plasma HDLc levels and decreased HDL percentiles (HDLc: controls = 1.23±0.26, mutation carriers = 0.83±0.37, mmol/L, p=0.0007; HDL%ile: controls = 41.8±21.2, mutation carriers = 17.3±19.2, p=0.004, n=23 controls and 12 carriers). No changes in LDLc, triglycerides or BMI were observed. Since mutations in ABCA8 reduce plasma HDLc levels, we assessed the role of ABCA8 in lipid efflux. Cholesterol efflux to lipid-free APOA-I was significantly reduced by 62.1% in fibroblasts from the proband with the E18-2 T>C mutation. In comparison, fibroblasts from heterozygous ABCA1 mutation carriers showed a 30-60% decrease in efflux to APOA-I depending on mutation severity. Wild type and P609R ABCA8 cDNA clones were generated from human liver RNA. A significant ~200% increase in cholesterol efflux to lipid free APOA-I was observed with wild type ABCA8, further suggesting that ABCA8 is a lipid transporter. A significant 75% decrease in the efflux of cholesterol to lipid-free APOA-I (p=0.02) was observed with the mutant P609R ABCA8 protein compared to wild-type. We show here that ABCA8 is a cholesterol transporter, and that mutations in ABCA8 are a novel cause for reduced plasma HDLc levels in humans.
Abstract 180: Myocardial Triacylglycerole Accumulation Is Related To BNP And Myocardial Redox State In Advanced Systolic Heart Failure
Authors: Benes Jan , Kazdova Ludmila , Malinska Hana , Pirk Jan , Spatenka Jaroslav , Kautzner Josef , Melenovsky Vojtech
Published: July 2021

PURPOSE:
Experimental studies suggest that accumulation of triacylglyceroles (TAG) in myocardium and ensuing lipotoxicity may contribute to the progression of heart failure (HF), but relevance to human HF remains uncertain. We strived to quantify myocardial TAG in HF patients and controls and to identify their determinants.

Methods:
49 myocardial samples from patients with terminal HF (52 yrs, 86% male, 45% CAD, 49% DCM, 6% other dg.) that underwent heart transplantation or LVAD implantation and 20 control samples from cadaverous organ donors without HF (42 years, 45% males) were analyzed. Myocardial TAG (determined chemically), reduced (GSH) and oxidized glutathione (GSSG) were determined.

Results:
Patients with HF had higher amount of myocardial TAG (8.07± 3.95 vs. 5.60±3.36 μmol/g wet tissue, p= 0.017) and lower levels of both GSH and GSSG (p< 0.05) with similar GSH/GSSG ratio. The amount of myocardial TAG was independent of age, gender, HF etiology, diabetes mellitus, BMI, mitral regurgitation grade and plasma lipids (total-, HDL-, LDL-cholesterol, TAG) but it correlated with plasmatic BNP level (p=0.049, r=0.28). In controls, a positive correlation between myocardial TAG and the myocardial redox state (GSH/GSSG ratio) was found (r=0.53, p=0.015). On the contrary, this correlation was negative (r= -0.31, p=0.02) in HF patients.

Conclusion:
Myocardial TAG in HF and controls have opposite relationship to redox state, indicating disparate mechanisms involved. Failing hearts display higher TAG accumulation, that is independent of comorbidities, but proportional to circulating BNP, supporting the role of natriuretic peptides in abnormal lipid homeostasis in HF.
Abstract 205: Recovery from Ischemia-Reperfusion Injury Is Metabolic Substrate-Dependent
Authors: Nadtochiy Sergiy M , Brookes Paul S
Published: July 2021

Introduction:
The adult heart utilizes mostly fat for energy production, with adaptation to different fuels (“metabolic plasticity”) being a hallmark of the healthy heart. However, metabolic maladaptation is known to occur in heart failure. As such, the ability of the heart to metabolize specific substrates could impact the outcome of pathological insults, such as ischemia-reperfusion (IR) injury. The aim of this study was to develop a system whereby adult mouse cardiomyocytes (AMC) subjected to IR injury could be supplied with different fuels, and metabolism measured in real-time.

Methods:
AMC were divided in 3 groups, supplied either with glucose (GLU, 5mM), palmitate/fat free BSA (FAT, 100µM) or GLU+FAT. A previously developed method for in-vitro IR injury using a Seahorse XF24 [1], was adopted for ACM. IR comprised 60 min. ischemia and 60 min. reperfusion, and additional metabolic parameters were measured separately using mitochondrial inhibitors and uncouplers [2]. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were simultaneously measured during the IR protocol, followed by a cell death assay.

Results:
FAT cells showed higher baseline OCR and lower ECAR rates compare to GLU cells, although uncoupled OCR was lower in FAT group, suggesting a lower metabolic reserve capacity for cells respiring on fat. Upon IR, the drop in pH was significantly greater in GLU compare to FAT, indicating faster lactate production. During reperfusion, both OCR and ECAR recovered to pre-ischemic levels in GLU cells but failed to do so in FAT cells. Post-IR cell death was significantly higher in FAT vs. GLU. Surprisingly, GLU+FAT (modeling a “physiologic” substrate mix) replicated the same metabolic profile and cell death as GLU.

Conclusions:
(i) AMC had better recovery from IR injury using glucose as fuel. (ii) Lower cell viability in FAT (vs. GLU) correlated with smaller metabolic reserve capacity and with a smaller pH drop during ischemia. This is consistent with a known protective role for acidification during IR injury. (iii) Mixed substrates (GLU+FAT) gave a similar response to glucose alone, suggesting that fat may not be toxic, rather glucose is protective, in IR injury.

[1] Circ Res. (2012), 110. 948-57.

[2] J Vis Exp. (2010), 46. pii: 2511.
Abstract 213: TRPV1 Mediates Remote and Direct Cardioprotection
Authors: Gross Eric R , Urban Travis J , Hsu Ana K , Qvit Nir , Gross Garrett J , Mochly-Rosen Daira
Published: July 2021

Introduction:
The transient receptor potential 1 channel (TRPV1) mediates signals from pain, heat, and/or noxious stimuli. TRPV1 sensitization can occur via a protein kinase C (PKC)-dependent mechanism in neurons. Therefore, we tested whether TRPV1 is a mediator of cardioprotection in models of ischemia-reperfusion and whether the molecular mechanism of cardioprotection occurs via PKC-induced TRPV1 channel sensitization.

Methods:
Male Sprague Dawley rats and H9C2 left ventricle-derived cells were used for whole animal and cellular ischemia-reperfusion studies to test this hypothesis. Statistical analysis regarding infarct size, calculated as percentage of area at risk per left ventricle, was performed by a one way ANOVA (*P<0.01).

Results:
Remote preconditioning-induced infarct size reduction
via
an abdominal surgical incision was blocked by prior administration of a selective TRPV1 peptide inhibitor, V1-B (3.0mg/kg), given over the incision site (Incision: 44±2*% V1-B+Incision: 65±2% versus Control: 64±1% n=6/group). Capsaicin (0.3mg/kg) given intravenously through the internal jugular vein reduced infarct size
in vivo
, which was blocked by prior capsazepine (TRPV1 inhibitor, 3.0mg/kg) administration (Capsaicin: 43±2* Capsaicin+ capsazepine: 64±4 versus Control: 62±3, n=7/group). Further in an
ex vivo
isolated heart model, infarct size reduction afforded by the selective epsilon PKC activator (pseudo epsilon RACK, 1uM) was partially blocked with prior treatment of V1-B (1uM), the TRPV1 peptide blocker (pseudo epsilon RACK: 20±2*%, pseudo epsilon RACK+V1-B: 42±4% versus control: 47±4%, n=7/group). TRPV1 expression was found in both whole heart homogenate and in the H9C2 cell line. Using a model of ischemia-reoxygenation in H9C2 cells, capsaicin treatment before and during ischemia-reoxygenation reduced cellular damage as assessed by MTT and LDH assays. Greater damage occurred with TRPV1 inhibition by capsazepine compared to control.

Conclusions:
Our studies suggest TRPV1 contributes an essential role for both remote and direct cardioprotection. Further studies are ongoing to determine the post-translational sites on TRPV1 and how a TRPV1-epsilon PKC protein-protein interaction induces cardioprotection.
Abstract 221: MitoROMK Expression Mitigates the Cellular Response to Oxidative Stress
Authors: Ho Alice S , Derungs Thomas , Foster D. Brian , O’Rourke Brian
Published: July 2021

We have recently reported that mitoROMK overexpression protects against, while knockdown of native ROMK exacerbates, oxidative stress-induced cell death and have proposed mitoROMK as the molecular correlate of the cardioprotective mitoKATP channel (Foster, Ho, et al Circ Res 2012). In order to better understand the mechanisms of protection, here we examined how alterations of ROMK expression affect glutathione redox responses during acute oxidative stress, utilizing cytoplasmic or mitochondrially targeted, genetically-encoded GSSG sensors. The tandem protein construct Grx1-roGFP2, a fusion of human glutaredoxin and redox sensitive GFP (Gutscher et al, Nat Methods 2008) was incorporated into adenoviral gene transfer vectors for expression in cardiac-derived H9C2 cells. Cells were subjected to treatment with the Complex III inhibitor Antimycin A (50µM) to induce endogenous mitochondrial reactive oxygen species (ROS) production, and changes in the glutathione redox potential were determined ratiometrically. MitoROMK overexpressing cells showed a significantly lower rate of mitochondrial matrix GSH oxidation secondary to H2O2 scavenging compared to both control and ROMK knockdown cell lines, indicating enhanced ability to maintain ROS balance. Increases in cytoplasmic GSSG were also blunted by mitoROMK overexpression. To determine if ROMK expression affected ROS-induced mitochondrial calcium overload, we also employed the mitochondrially-targeted ratiometric Ca2+ probe GEM-GECO (Zhao et al Science 2011). The same Antimycin A challenge led to large mitochondrial Ca2+ increase in control and knockdown cell lines, but this response was significantly suppressed in mitoROMK overexpressing cells. This could not be explained by differences in baseline mitochondrial membrane potential (ΔΨm; determined using the potentiometric probe tetramethylrhodamine methyl ester), because both control cells and ROMK overexpressors showed similar ΔΨm, while ROMK knockdown cells were more depolarized. The results suggest that enhanced cell survival in mitoROMK overexpressing cells is mediated by improved mitochondrial ROS balance and calcium handling, providing a key molecular link between mitochondrial K+ channels and cardioprotection.
Abstract 245: Cardiac Stem Cells and Myocyte Regeneration in Diabetic Cardiomyopathy
Authors: Ogorek Barbara , Matsuda Alex , Wybieralska Ewa , Kostyla James , Borghetti Giulia , Caballero Maria Virginia , Canata Antonio , Sorrentino Andrea , Leri Annarosa , Anversa Piero , Rota Marcello , Kajstura Jan
Published: July 2021

The mammalian heart contains a pool of resident c-kit-positive cardiac stem cells (CSCs), raising questions concerning their role in the etiology of the diabetic myopathy. The objective of the current study was to determine whether the negative effects of diabetes on the adult heart are dictated by defects in CSC growth and lineage commitment. Type I insulin-dependent diabetes mellitus was induced in mice by streptozotocin administration. The kinetics of CSCs and cardiomyocytes was measured 3, 7, 10, 20, and 30 days after the onset of diabetes, by implementing a hierarchically structured cell system, which allows the quantitative analysis of the rate of cell turnover. The multiple variables required to apply this mathematical model were measured by confocal microscopy. They included first the number of CSCs, LCCs (lineage committed cells: myocyte progenitors-precursors), transit amplifying myocytes, and post-mitotic myocytes in the left ventricular (LV) myocardium. Additionally, the fraction of cycling CSCs and the length of their cell cycle were measured, together with the number of cardiomyocytes dying by apoptosis and necrosis. The specificity of labeling was documented by spectral analysis. The diabetic heart was characterized by a severe time-dependent loss in LV post-mitotic myocytes, dictated primarily by a defect in cell renewal. Under control conditions, 20% of myocytes were replaced per month in the adult mouse heart through activation, cell cycle reentry, and differentiation of CSCs. This value decreased sharply to 6%, 1.4%, 0.5%, 0.07%, and 0.05% at 3, 7, 10, 20 and 30 days after the administration of streptozotocin. The myocardium showed a progressive increase in old cardiomyocytes expressing the senescence-associated protein p16INK4a and p53. Importantly, the severe impairment in myocyte regeneration was coupled with an increase in LV end-diastolic pressure, and a decrease in LV systolic pressure, LV developed pressure, positive and negative dP/dt. Thus, our data indicate that the diabetic myopathy has to be viewed as a stem cell disease in which the decrease in the number of myocytes is a secondary event, resulting from defects in replication and lineage specification of cardiac stem cells.