bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P004: Cardiac Regeneration in Neonatal Mice Following Ischemic Injury
Authors: Porrello Enzo R , Mahmoud Ahmed I , Simpson Emma , Hill Joseph A , Richardson James A , Olson Eric N , Sadek Hesham A
Published: March 2022

As a consequence of the adult mammalian heart's limited regenerative capacity, ischemic heart disease is the leading cause of morbidity and mortality in the developed world. We have recently identified a brief window during post-natal development when the mammalian heart retains significant cardiac regenerative potential. Similar to adult zebrafish, the 1 day-old neonatal mouse heart is capable of complete cardiac regeneration following amputation of the entire ventricular apex. One major unresolved question is whether the neonatal mouse heart can also regenerate in response to myocardial ischemia. Here, we demonstrate that the neonatal mouse heart is also capable of undergoing a cardiac regenerative response to replace lost cardiomyocytes following myocardial ischemia. Following permanent ligation of the left anterior descending (LAD) coronary artery of 1 day-old neonatal mice, we documented evidence of significant cardiac injury and myocyte death by histology and echocardiography at 1 and 4 days after LAD ligation (Ejection Fraction (MI vs. Sham) = 70.9±10.1% vs. 98.6±0.3%, P<0.05). Remarkably, the 1 day-old neonatal mouse heart completely regenerated the necrotic myocardium within 21 days, with minimal fibrosis and full recovery of cardiac function (Ejection Fraction (MI vs. Sham) = 94.6±1.4% vs. 97.9±0.1%). Similar to the regenerative response of neonatal mice to apical resection, the regenerative response of the neonatal mouse heart to ischemic injury was associated with robust and widespread activation of cardiomyocyte proliferation (∼11-fold), which peaked at day 7 post-injury. In contrast, following LAD ligation at 7 days-of-age, the cardiac regenerative response of neonatal mice was significantly impaired and by 14 days-of-age, the cardiac injury response was characterized by a fibrotic repair process that was reminiscent of the adult heart's response to ischemic injury. Additionally, over-expression of miR-195, a potent inhibitor of cardiomyocyte proliferation, impaired the cardiac regenerative response of neonatal mice. Thus, the neonatal mammalian heart is capable of complete regeneration following ischemic myocardial necrosis and cardiomyocyte proliferation appears to play an important role in this process.
Abstract P012: KDR Expression Enhances Commitment Toward the Cardiac Lineage of hESC- and iPS-Derived Cd15
+
Progenitors
Authors: Di Pasquale Elisa , Paola Portararo , Rizzi Roberto , Mancino Monica , Condorelli Gianluigi
Published: March 2022

Cell-based therapies represent exciting therapeutic options for myocardial regeneration after ischemic injury. Although many cell types have been proposed as a source of cardiac myocytes, the type of progenitor as well as the optimal conditions for their stimulation still represent major challenges. Moreover, molecular characterization of cardiac progenitors (CP)
in vitro
is still unsatisfactory: the identification of novel surface markers for their selective isolation is of utmost importance. In our study we employed different human ESCs and induced pluripotent stem (iPS) cells lines to set up a simple, reliable and efficient protocol to selectively induce cardiac fate
in vitro.
At first, we comparatively applied different methods and monitored the differentiation process by FACS analysis, RT-PCR and immunofluorescence for early and late markers. Our results show that sequential treatments with ActivinA and BMP4 or BMP2, ascorbic acid and TGFβ1 in a specific media formulation induce CD15
+
cardiac progenitor population with an enhanced cell vitality compared to other methods. While we confirmed that CD15 is among the earliest markers upregulated after differentiation, the definition of other associated markers could better specify progenitors committed toward cardiomyogenesis. For this purpose, we systematically evaluated induction of other markers already shown associated with cardiac differentiation or heart development, that is PDGFRα, KDR, Sca1, CXCR4, cKIT. Our result demonstrate that a specific population expressing both KDR and CD15 surface markers is more committed toward cardiovascular lineages. The induced KDR
+
/CD15
+
cell population exhibits higher levels of Gata4 and Isl1 expression (2-fold induction), compared to the KDR
−
/CD15
+
counterpart and, after isolation, gives rise to 66.15% Troponin I positive cells (43 out of 65 in a single representative experiment), against the 26.5% (18 out of 68) detectable in the KDR
−
/CD15
+
population. In conclusion, our studies, though preliminary, strongly indicate that KDR in CD15
+
progenitor cells is an earlier marker of cardiomyogenesis.
Abstract P029: Resolving Discordance of Human Cardiomyogenesis Estimates in the Healthy Adult Heart
Authors: Elser Jeremy , Margulies Kenneth
Published: March 2022

Rationale:
Estimates of the endogenous cell turnover rates in healthy human hearts conflict, varying from 0% to over 30%. Turnover rate age-dependence is also controversial with some studies indicating decreasing turnover with advancing age and other studies suggesting that turnover increases with age.

Methods/Results:
We created a hybrid mathematical model encompassing two prominent analyses of human adult cardiomyogenesis that concluded highly disparate turnover rates. The model was programmed to accept input parameters for age-dependent cardiomyogenesis and apoptosis. High-turnover input parameters (10%–30%, increasing with age), extracted from a recent publication by Kajstura, et. al., were used to create 12 model patient hearts with ages and birthdates corresponding to the 12 patients analyzed by a C14 fate-mapping technique in a publication by Bergmann, et. al., which reported low turnover rates of 1%–0.45%, decreasing with age. Modeled hearts parameterized with high, age-increasing input turnovers produced low (5%–0.5%), age-decreasing estimates of turnover when the C14 analysis technique was applied. This systematic underestimation reflects the inability of fate-mapping models to account for turnover events in which myocytes created after patient birth are destroyed prior to patient death. This effect may underestimate true turnover events by over 90% and is more severe for patients with longer lifespans, predisposing such techniques to report decreasing turnover with advancing age. We next evaluated the effect of input parameter uncertainty on the hybrid model. We observed non-linear sensitivity of fate-mapping algorithms to polyploidization correction factors, with 20% variation in correction factor leading to approximately 30% changes in reported turnover. The Kajstura model was acutely sensitive to estimates of myocyte half-life (Δ20% produced a 45% change in average myocyte age) and stem cell expansion coefficient (Δ20% produced a 2-fold change in reported turnover).

Conclusions:
Accounting for short-lived myocytes in fate-mapping models reconciles apparent differences between the models. Recognition of realistic input parameter uncertainty improves interpretation of these models.
Abstract P037: Viable Autologous Umbilical Cord Patch for Aortic Reconstruction in Newborn Piglets
Authors: Hammel James M , Lang Haili , Siecke Rebecca , Radio Stan
Published: March 2022

Background:
Aortic surgery in neonates often uses a patch of cryopreserved allogeneic artery. Drawbacks include alloimmunization in infants who may later need heart transplant. We hypothesized that the umbilical cord (UC) may be a non-immunogenic, viable vascular patch suited to neonatal aortic surgery. This was evaluated in ex vivo studies of human UC, and in vivo study in piglets.

Method:
Human UC (20) were collected after vaginal birth, were dilated and measured. Swab cultures were taken. For final culture UC was put in Thiol, and held for 4 wk. Disinfection was in dilute hypochlorite for 30 sec to 5 min. UC segments were stored in HTK solution with antibiotic/antifungal agent at 4 deg for 3 to 9 d. Samples were fixed for histology. At 9 d, samples were put in tissue culture to check viability. Then, 18 farm piglets in three litters were delivered by hysterotomy. Each UC was sanitized, cultured, and stored as described for human UC. At 7 d, each piglet underwent laparotomy. A defect made in the abdominal aorta was patched with autologous UC or with PTFE. Piglets were killed after 8 and 12 wk to examine the patched aorta.

Result:
Human UC diameter was 8-11 mm. Cultures of 5 UC in Dakins for 30 sec, 1, 2, 3, and 4 min were all negative. All later UC were treated for 30 sec only. One culture from one UC segment after 9 d was positive; final culture from this UC wwas negative. Histology did not differ at 9 d from day of delivery (n=3). Cells grew from 9 d UC in tissue culture (n=2). Piglet UC at 7 d also held viable cells. Piglet UC patch worked well in surgery. Hemostasis was rapid with UC, and was longer with PTFE. Three piglets were killed before 8 wk due to hernia. There was no dehiscence, infection, or aneurysm in either group. UC-patched aorta lengthened 114%, PTFE grew 21%. Patched aorta was non-stenotic in both groups, 92% of normal in UC vs. 95% in PTFE. By histology the UC did not remain viable, but calcified and broke into fragments. Beneath both UC and PTFE patches, fibroblast proliferation spanned the aortic defect. In UC piglets, this layer enlarged after UC fragmentation.

Conclusion:
UC can be sterilized and kept viable after vaginal birth . UC patch worked well for repair of an aortic defect in piglets, but UC viability and growth were not seen. UC needs further study before trial in human aortic surgery.
Abstract P062: Loss of Plasma Membrane Integrity Occurs in 2 Stages During Early Myocardial Ischemia/Reperfusion Injury
Authors: Zhang Ming , Charlagorla Pradeepkumar , Liu Junying , Balroop Javi , Ko Eun Hee , Green Danielle , Shaka-Idusuyi Oghomwen , Patel Monaliben , Richardson Arlan , Rushbrook Julie
Published: March 2022

Myocardial ischemia is an acute health emergency. Reperfusion, critical for salvaging ischemic tissues, contributes to the acute tissue damage known as ischemia-reperfusion (I/R) injury. While therapeutic approaches aimed at reducing I/R injury have been promising in preclinical studies, clinical studies have been inconclusive, suggesting an inadequate knowledge of the mechanisms underlying I/R injury and their time-frame. In the current work, myocardial necrosis was evaluated in a murine I/R model using a propidium iodide-based fluorescent method that evaluated loss of plasma membrane integrity (LPMI) during the first 24 hours of reperfusion and permitted histological analysis of other pathological events on adjacent sections. LPMI was found to develop in two stages in WT animals: a significant increase occurred between 0 and 1 hour of reperfusion and was maintained through 6 hours; an additional increase was apparent at 24 hours. Complement C3 deposition lagged LPMI by 2 hours and C3
−/−
mice were protected from second-stage LPMI but not first-stage. Transgenic mice overexpressing the anti-oxidant enzymes superoxide dismutase or catalase were protected from first stage LPMI but not second stage. The results provide support for the development of therapeutic approaches that are directed at the early stages of reperfusion and take into account the two phases of ischemia-related myocardial damage, designing strategies targeting the reactive oxygen species of first stage and the complement-mediated events of second stage.
Abstract P084: Cardiac-Specific Overexpression of Human Histidine-Rich Calcium Binding Protein (HRC-S96A) Genetic Variant Impairs Sarcoplasmic Ca
2+
Handling, Resulting in Arrhythmias
Authors: Singh Vivek P , Cai Wenfeng , Dong Min , Liang Qian , Lam Chi Keung , Gao Xiaoqian , Chen Shan , Arvanitis Demetrios A , Haghighi Kobra , Wang Hong S , Kim Do H , Cho Chunghee , Kranias Evangelia G
Published: March 2022

Disturbed Ca-homeostasis in the cardiomyocyte is a hallmark of human and experimental heart failure. The histidine-rich Ca binding protein (HRC), an intraluminal SR protein is an additional regulator of SR Ca2+ cycling. Previous biochemical studies have shown that HRC can bind to triadin (a component of ryanodine receptors complex) and SERCA2a, suggesting that HRC may be involved in the regulation of SR Ca2+ release and Ca2+ uptake. We recently identified a human HRC genetic variant (S96A), which appears to correlate with ventricular arrhythmias and sudden death in dilated cardiomyopathy (DCM) patients. To gain insight into the physiological and pathological significance of the S96A-HRC variant, we generated transgenic mice with cardiac-specific overexpression of human HRC wild-type (HRC-WT) and S96A mutant (HRC-S96A) in the HRC null (HRC KO) background. We obtained lines with similar expression levels of HRC-S96A and HRC-WT for further characterization. Overexpression of human HRC-S96A resulted in decreased fractional shortening by 22% (7.7 ± 0.5% in HRC-WTs vs. 6.0 ± 0.4% in HRC-S96A), rates of contraction by 20% (75 ± 5 µm/sec in HRC-WTs vs. 60 ± 4 µm/sec in HRC-S96A) and rates of relaxation by 20% (87 ± 6 µm/sec in HRC-WTs vs. 70 ± 4.5 µm/sec in HRC-S96A) compared with HRC-WT. Myocytes isolated from HRC-S96A mice had diminished Ca2+ transient amplitude and delayed half-decay time of the Ca2+ transient. In addition, the frequency of Ca2+ sparks was significantly higher although caffeine-induced SR Ca2+ release (SR load) was reduced in HRC-S96A cells. To determine the effect of S96A-HRC under stress conditions, 5 Hz field stimulation in the presence of 1 µmol/L Isoproterenol was applied: after-contractions were developed in 46% (16 of 35) of HRC-S96A cardiomyocytes, compared to 12% (4 of 33) of HRC-WTs mice. The findings of the present study demonstrate that the human HRC-S96A variant results in impaired myocytes Ca2+ handling, associated with depressed SR Ca2+-uptake rate and increased rate of SR Ca2+ leak, which may destabilize the cells, promoting aftercontractions under stress conditions. Thus, there appears to be a link between this genetic variant and ventricular arrhythmias in DCM human carriers.
Abstract P101: Genetic Variation of Phenylethanolamine-N-methyltransferase Influences Cardiovascular Function in Healthy Humans
Authors: Mohammed Abdulaziz A , Wheatley Courtney M , Cassuto Nicholas A , Snyder Eric M
Published: March 2022

Objectives:
Genetic susceptibility to hypertension can be caused by alterations in cardiovascular and/or renal function. Previous work using genome-wide association studies have demonstrated that the gene that encodes phenylethanolamine-N-methyltransferase (PNMT) is a candidate for hypertension. Follow-up studies confirmed that the gene that encodes PNMT may alter susceptibility to hypertension in African Americans, although no mechanistic data was assessed. Genetic variation of PNMT alters the catecholamine response to exercise, and variation of PNMT at position -182 has been associated with susceptibility to multiple sclerosis.

Methods:
We sought to determine the influence of genetic variation of PNMT (A-182G) on renal Na
+
handling and cardiovascular function in humans. We collected serum epinephrine (Epi) and norepinephrine (NE), 24-hour urine, serum Na
+
, K
+
, and creatinine, and measured cardiac output (Q, acetylene rebreathing), heart rate (HR, 12-lead EKG), blood pressure (systolic, SBP, diastolic, DBP, and calculated mean arterial, MAP) and calculated fractional excretion of Na
+
(F
E
Na), stroke volume (SV), and systemic vascular resistance (SVR) in 20 normotensive subjects (n: AA=6 and GG=14).

Results:
We found that the GG genotype had higher Epi and Epi/NE when compared to AA (Epi=98±21 vs. 53±10pg/l; NE= 344±56 vs. 462±79pg/l; Epi/NE= 0.31±0.07 vs. 0.13±0.03, for GG and AA, respectively, mean±SE, p<0.05 for Epi and Epi/NE). We found no differences between the genotype groups in 24hr. renal Na
+
handling (serum Na
+
, urine Na
+
, or F
E
Na). We found no differences in HR, SBP, or SV between the groups. The GG group had a trend towards a lower DBP, and MAP, but these did not reach statistical significance (DBP= 70±3 vs. 75±2mmHg; MAP= 83±3 vs. 87±2mmHg, for GG and AA, respectively, mean±SE). The GG group had a higher Q and a lower SVR when compared to the AA group (Q= 6.0±0.5 vs. 4.8±0.4 l/min; SVR= 1020±64 vs. 1316±95 dynes*sec/cm
5
, for GG and AA, respectively mean±SE, p<0.05).

Conclusion:
These results suggest that genetic variation of PNMT can influence resting blood pressure in normotensive individuals and that this may be the result of cardiovascular function, rather than renal Na
+
handling.
Abstract P113: Protein Tyrosine Phosphatase-Like A Is a Unique Regulator for Myogenesis
Authors: Lin Xi , Yang Xiangsheng , Li Qi , Ma Yanlin , Lin Xia , Chang Jiang
Published: March 2022

Background:

Protein tyrosine phosphatase-like A (PTPLa) is highly expressed in skeletal and cardiac muscle. Therefore, it has been implicated to play a role in skeletal myogenesis and cardiogenesis. Mutations in PTPLa correlated with arrhythmogenic right ventricular dysplasia in humans and congenital centronuclear myopathy with severe hypotonia in dogs. However, the molecular mechanisms of PTPLa in myogenesis are unknown.

Method and results:

Mouse myoblast C2C12 cells were used to generate PTPLa deficient and PTPLa overexpression cell lines and the cells were cultured in proliferating and differentiating media, respectively, to study regulatory role of PTPLa in cell proliferation and differentiation. We demonstrated that PTPLa was required for myoblast growth and differentiation. The cells lacking PTPLa remained immature and failed to differentiate into mature myotubes along with repressed MyoG expression. To define the role of MyoG in PTPLa-mediated myoblast differentiation, myoblast cell line lacking MyoG expression was generated. Two cell lines, the PTPLa-deficient and MyoG-deficient myoblasts were used to demonstrate that PTPLa-mediated myoblast differentiation was through MyoG regulation. Meanwhile, the impeded cell growth with an obvious S-phase arrest was observed in PTPLa-deficient myoblasts. Further study demonstrated that the upregulation of cyclin D1 and cyclin E2 complexes along with a compromised Cdk1 activity contributed to the mutant cell S-phase arrest and eventually led to the retarded cell growth. Finally, the transcriptional regulation of PTPLa gene was explored. We identified that PTPLa was a new target gene of serum response factor (SRF). Skeletal- and cardiac-specific SRF knockouts resulted in significant decreases in PTPLa expression, suggesting a conserved transcriptional regulation of PTPLa gene in mice.

Conclusion:

PTPLa is a unique pro-myogenic factor regulated by SRF. It regulates myoblast proliferation and differentiation through MyoG and cell cycling signaling pathway.
Abstract P160: N-Type Ca
2+
Channel Blockade Prevents Sudden Death in Mice with Heart Failure
Authors: Yamada Yuko , Kuwahara Koichiro , Kinoshita Hideyuki , Kuwabara Yoshihiro , Nakagawa Yasuaki , Shibata Junko , Minami Takeya , Yasuno Shinji , Usami Satoshi , Nishikimi Toshio , Ueshima Kenji , Mori Yasuo , Nakao Kazuwa
Published: March 2022

Background
- Increased sympathetic nerve activity is involved in the increased arrhythmogenicity seen in patients with chronic heart failure. N-type Ca
2+
channel (NCC) modulates sympathetic nerve activity by regulating calcium entry at sympathetic nerve terminal, which triggers the release of the neurotransmitter noradrenalin. The ability of NCC blockade to prevent lethal arrhythmias associated with heart failure has never been tested, however.

Methods and Results
- We compared effects of cilnidipine (Cil), dual N-and L-type Ca
2+
channel blocker, with those of nitrendipine (Nit), a selective L-type Ca
2+
channel blocker, in a cardiac-specific, dominant-negative form of neuron-restrictive silencer factor transgenic mice (dnNRSF-Tg), a mouse model of dilated cardiomyopathy leading to sudden arrhythmic death. Hemodynamic and echocardiographic analyses showed no significant difference among the control, Cil, and Nit groups. In contrast, Cil dramatically improved the survival rate (p<0.001) and reduced ventricular arrhythmia (p<0.05) among dnNRSF-Tg mice, compared with control vehicle or Nit (n=22 for WT, n=20 for Tg without drugs, n=22 for Tg with Cil, n=23 for Tg with Nit). Cil improved the imbalance in autonomic nerve activity assessed by heart rate valiability, and also reduced the increased urinary excretion of noradrenalin seen in dnNRSF-Tg, whereas Nit did not.

Conclusions
- NCC blockade ameliorated disturbed autonomic nerve function and improved survival in a mouse model of dilated cardiomyopathy and sudden death. Our findings suggest NCC blockade is a potentially useful approach to preventing sudden cardiac death in patients with heart failure.
Abstract P184: Rescue of Reductive Stress by Nrf2 Deficiency in Protein Aggregation Cardiomyopathy Mice
Authors: Namakkal Soorappan Rajasekaran , Khanderao Gayatri D , Miller Corey J , Olson Curtis , Rajasekaran Vasanthi , Kannan Sankaranarayanan , Litwin Sheldon E , Davidson Christopher J
Published: March 2022

Background:
We recently discovered that the overexpression of human mutant αB-crystallin (hR120GCryAB) in mouse resulted in protein aggregation cardiomyopathy (PAC) and reductive stress (RS) in the heart tissue (Rajasekaran et.al Cell, 2007). Further, we identified that sustained activation of Nrf2 (nuclear erythroid 2 related factor 2)/ARE signaling as a causal mechanism for RS in the MPAC mice. We hypothesize that abolishing Nrf2 could prevent RS in R120GTG mice and thereby rescue them from cardiomyopathy and heart failure.

Methods:
To elucidate loss-of-function mechanisms for Nrf2 on RS and MPAC, we produced (1) NTG/WT, (2) R120GTG/WT, (3) R120GTG/Nrf2+/- and (4) R120GTG/Nrf2-/- mice by intercrossing R120GTG X Nrf2-KO. Primarily, we determined the rate of survival and performed echocardiography to understand the degree of pathogenesis and cardiac function over time (n=6-10). Next, we analyzed the redox state and protein/mRNA expression for major antioxidants including enzymes that are involved in glutathione metabolism in the heart.

Results:
Disruption of Nrf2 prolonged the survival of MPAC mice along with no signs of cardiac dysfunction (by ECHO). The R120GTG mice with Nrf2 deficiency (either Nrf2+/- or Nrf2-/-; n=4) had significantly decreased glutathione (but equal to NTG) levels when compared with R120GTG/WT cohorts. Such a decline in GSH resulted in quenching of “reducing power” to prevent “reductive stress” and facilitated redox homeostasis in the myocardium. Interestingly, the intercross mice showed no cardiac hypertrophy even at 12 months while the R120GTG exhibited 90% mortality due to PAC/heart failure. Further, protein and mRNA levels for major antioxidants were found to be normal in the R120GTG/Nrf2+/- while there was significant down regulation of these parameters in the R120GTG/Nrf2-/- mice when compared to NTG/WT suggesting that Nrf2-independent pathways are inadequate to maintain the antioxidant capacity under stressed conditions.

Conclusions:
These results demonstrate a critical role for Nrf2 underlying antioxidant potential in the myocardium. Abrogating RS via Nrf2 might be highly relevant to cure the protein aggregation diseases that are coupled with elevated intracellular reducing potential.
Abstract P188: Suppression of MK2 Signaling Protects Against Pressure Overload-Induced Cardiac Dysfunction
Authors: Khairallah Maya , Dingar Dharmendra , Khairallah Ramzi J , Vaniotis George , Shi Yanfen , Gaestel Matthias , Stanley William C , Tardif Jean C , Allen Bruce G
Published: March 2022

p38 mitogen-activated protein kinases (MAPKs) regulate a broad range of cellular activities but have been implicated in the pathogenesis of cardiovascular diseases. The mechanisms whereby p38 exerts divergent effects are unknown. Current p38 inhibitors both produce serious side effects and loose efficacy with chronic use. Hence, a better understanding of the downstream targets of p38, i.e. MAPK-activated protein kinases (MKs), is essential. We hypothesized that inhibition of MK2 activity (MK2-/-) during chronic pressure overload is cardioprotective. Twelve-week-old MK2-/- and littermate control (MK2+/+) mice were subjected to transverse aortic constriction (2 wks, n=8). MK2-/- mice showed a ∼20% reduced increase in heart weight-to-tibia length ratio (Fig A) despite similar increases in mean blood pressure and ANP expression. Compared to controls, banded MK2-/- hearts also showed preserved left ventricular: (i) fractional shortening, (ii) ejection fraction (preserved at 72.5 ± 2.2% after banding for MK2-/- but decreased from 67.9 ± 3.6% to 58.8 ± 3.4% in MK2+/+, p<0.05, Fig B), and (iii) diastolic function evidenced by the ratio of transmitral and myocardial early diastolic velocities (E/Em preserved after banding in MK2-/- but increased by 78.8 ± 11.1% in MK2+/+, p<0.01). MK2 deficiency did not reduce interstitial fibrosis. For equivalent respiration rates, mitochondria from MK2-/- hearts showed a significant decrease in Ca2+-sensitivity for mitochondrial permeability transition pore opening (p<0.05, Fig C). Overall, these results suggest that MK2 mediates part of the detrimental remodeling evoked by chronic pressure overload-induced activation of p38.
Abstract P211: MRP4: A Novel Protein Involved in Camp Homeostasis in the Heart
Authors: Abi-Gerges Aniella , Sassi Yassine , Mougenot Nathalie , Jacquet Adeline , Engelhardt Stefan , Hatem Stéphane N , Hulot Jean-Sébastien , Lompré Anne-Marie
Published: March 2022

Rationale:
Transporters of the
ABCC
family, called MRP4 and MRP5, have been described to control cAMP homeostasis by extruding it from various cell types.

Objective:
The aim of our study was to determine the role of MRP4 in cAMP homeostasis and heart function and its impact on cardiac hypertrophy (CH).

Methods and results:
MRP4 KO mice and MRP4 KO with cardiac overexpression of the HCN-based cAMP sensor were used. Echocardiography and morphology studies showed that 3 month-old MRP4 KO mice displayed normal cardiac function (FS: 35.7% ± 1.02 in KO vs 36% ± 1.97 in WT) and cardiac phenotype (HW/TL: 7.59 ± 0.14 in WT mice vs 7.75 ± 0.15 in KO) in basal condition. Stimulation of WT and MRP4 KO mice by increasing concentrations of isoproterenol (ISO: 0.1μg/kg to 3 mg/kg) induced dose-dependent positive inotropic and chronotropic effects but there was no difference in the rate of contraction (Vmax: 11214 ± 682 in WT vs 10697 ± 691 in KO) or in the heart rate (724 ± 21 in WT vs 711 ± 21 in KO) between both groups. Cardiac myocytes were isolated from WT and MRP4 KO mice with cardiac expression of the HCN-based FRET sensor and intracellular cAMP concentrations were measured by FRET. Application of ISO (1nM) induced a similar increase in cAMP level in WT and MRP4 KO mice (YFP/CFP: 4.4 ± 0.15 in WT vs 4.1 ± 0.44 in KO cells), whereas cAMP signal was greater in MRP4 KO compared to WT mice after PDE inhibition by IBMX (ISO 1nM + IBMX 300µM: 8.1 ± 0.6 in WT vs 11.8 ± 0.62 in KO cells; p<0.01). PDE3A and PDE4A mRNA level was 1.6 fold higher in the myocardium of MRP4 KO compared to WT mice. MRP4 KO mice displayed age-dependent CH (HW/TL in 9 month-old mice: 9.8 ± 0.57 in KO vs 7.82 ± 0.13 in WT mice; p<0.01). 3 month-old MRP4 KO and WT mice were subjected to thoracic aortic clamping (TAC) or sham operated (Sh) and CH was assessed at 12 weeks after the surgery by echocardiography and morphometric studies. Heart function was also examined by hemodynamic technique in basal conditions and upon a β-adrenergic stimulation in WT (Sh and TAC) and MRP4 KO (Sh and TAC) mice. Alteration of left ventricular dP/dt upon β-adrenergic stimulation after TAC was more severe in MRP4 KO compared to WT mice.

Conclusion:
Taken together, our results show that both PDEs and MRP4 are important in cAMP homeostasis.
Abstract P228: MicroRNA Expression in Hypertension and Left Ventricular Hypertrophy in Rat
Authors: Kain David , Yagil Chana , Landa-Rouben Nathalie , Jacob-Hirsch Jasmine , Dor Chen , Yagil Yoram , Leor Jonathan
Published: March 2022

Background:
Heart injury and stress can cause a hypertrophic growth response in cardiac myocytes, which is characterized by an increase in cell size, enhanced protein synthesis, and activation of fetal genes, which can lead to heart failure (HF) and death. Given the emerging roles of microRNAs (miRNAs) in modulation of cellular phenotypes, we aimed to investigate miRNAs that are regulated during hypertension, left ventricular hypertrophy (LVH) and HF by using a rat model.

Methods and Results:
Male salt-sensitive (SBH/y) and salt-resistant (SBN/y) Sabra rats were obtained from the Israeli Rat Genome Center. Animals were salt-loaded with deoxycorticosterone-acetate (75 mg DOCA pellet s.c.) and 1% NaCl in drinking water for 16 weeks. Systolic blood pressure was determined by the tail-cuff method. Cardiac images were obtained using cardiac MRI using a specific small animal imaging protocol. Rats were sacrificed at different time points. miRNA expression was studied in the left ventricle using GeneChip miRNA Array (affymetrix). Expression of miRNAs related to cardiac hypertrophy or cell proliferation, including miR-132, miR-212, miR-29, miR-31and miR-205, increased more than 1.5 fold in salt-loaded SBH/y compared with salt-loaded SBN/y. In contrast, expression of MiR-182, miR-17-3p and miR-130b, known to be down-regulated in heart disease, diminished more than -1.5 fold in salt-loaded SBH/y compared with salt-loaded SBN/y.

Conclusions:
In animal model of salt-sensitive hypertension and LVH, up- and down-regulation of distinct miRNAs in the heart suggest novel therapeutic targets to prevent and reverse LVH and HF.
Abstract P235: Endothelium-Specific Prokineticin Receptor 1 Inactivation in Mice Leads to Endothelial Dysfunction and Metabolic Syndrome
Authors: Nebigil Canan G
Published: March 2022

Reciprocal relationships between endothelial dysfunction and insulin resistance result in a vicious cycle, linking between cardiovascular and metabolic disorders. Here we provide the first evidence that endothelial specific loss of prokineticin receptor-1 (PKR1) in mice leads to endothelial dysfunction and low capillary formation, ultimately contributing cardiovascular, kidney disorders and development of insulin resistance. Endothelial cells derived from these mutant hearts displayed an impaired proliferation rate and vessel-like formation. Low capillary formation in mutant heart, kidney, pancreas and adipose tissues was evident. The mutant aortas displayed increased collagen deposition, fenestrated endothelium with junctional defects, and impaired relaxation due to nitric oxide synthesize-deficits without altering blood pressure. Mutant hearts have thin ventricular wall due to an increase in apoptotic cardiomyocytes. Mutant kidneys have abnormal glomerulus structure with an increase in apoptotic cell numbers and phosphate excretion. Mutant mice exhibited low fasting blood glucose and high serum insulin levels. They cleared the blood glucose at a considerably slower rate than control mice accompanied with impaired insulin tolerance. The insulin resistance induces compensatory β cell hypertrophy but not expansion due to impaired survival signaling. Induction of cell death and fibrosis is induced by hypoxia in mutant adipocytes leads to increase in HIF1a and collagen expression. Moreover, an increase in serum FFA and inhibition of insulin-signaling pathway in adipocytes may represent an early event in systemic insulin resistance. This mutation provides a genetic model for metabolic syndrome and should facilitate studies of both the pathogenesis and therapy of cardiovascular, renal and metabolic disorders in humans.
Abstract P240: Mitochondrial Reactive Oxygen Species Regulate the Cardiac Na
+
Channel in Heart Failure
Authors: Liu Man , Liu Hong , Jeong Euy-Myoung , Gu Lianzhi , Dudley Samuel C
Published: March 2022

Background:
Previously, we have shown that elevated intracellular NADH causes a decrease in cardiac Na
+
current (

I
Na

) signaled by an increase in mitochondrial reactive oxygen species (ROS). The decrease in

I
Na

can be ameliorated by NAD
+
or a mitochondrial specific anti-oxidant, mitoTEMPO. It is known that cardiomyopathy is associated with reduced

I
Na

. Therefore, we tested whether the NADH-mitochondrial ROS pathway was involved in the reduction in

I
Na

in cardiomyopathic mice.

Methods:
Nonischemic cardiomyopathy was induced in C57BL/6 mice 6 weeks after unilateral nephrectomy, deoxycorticosterone acetate (DOCA) pellet implantation, and salt water substitution. Sham operated mice were used as controls. Ventricular myocytes isolated from mice were utilized for whole-cell patch clamp recording, intracellular NADH/NAD
+
level measurements, and mitochondrial ROS monitoring with confocal microscopy.

Results:
Compared to the sham mice, the left ventricular volume was significantly enlarged (105.5 ± 4.4 µL vs. 91.6 ± 4.0 µL, P<0.05), and the ejection fraction was significantly reduced (39.2 ± 1.6% vs. 48.7 ± 1.6%, P<0.05) in DOCA mice. Intracellular NADH level was increased (2.78-fold; P<0.01), and

I
Na

was decreased (62±8%; P<0.01) in myocytes of DOCA mice vs. sham. NAD
+
(500 μ M) and mitoTEMPO (10 μ M) recovered

I
Na

(90±9% and 103±9% of sham, respectively, P>0.05). Mitochondrial ROS overproduction was observed with DOCA mice myocytes by MitoSOX Red (∼4.8-fold of sham; P<0.01). NAD
+
and mitoTEMPO decreased ROS in DOCA mice myocytes (∼20±6% of DOCA for both, P<0.01).

Conclusions:
NADH and mitochondrial ROS were elevated, and

I
Na

was decreased in nonischemic cardiomyopathy. Maneuvers that reduced mitochondrial ROS restored

I
Na

. Since reduced

I
Na

and the subsequent slow conduction velocity are thought to contribute to arrhythmic risk in this condition, NAD
+
and mitochondrial anti-oxidants may have anti-arrhythmic activity in cardiomyopathy.
Abstract P243: Simultaneous Decrease in Glucose Uptake, Increase in Glucose Oxidation, and Improved Contractile Function by Hearts from Sucrose-Fed Rats
Authors: Harmancey Romain , Lam Truong , Lubrano Genna , Vela Deborah , Taegtmeyer Heinrich
Published: March 2022

The heart responds to acute hemodynamic stress by increasing carbohydrate oxidation. This metabolic response is impaired in diabetes when excess fatty acid availability inhibits glucose oxidation (Randle's “glucose-fatty acid cycle”). Impaired glucose uptake and oxidation are believed to result in energy deficiency and poor contractile function. We now propose that in the heart insulin resistance, defined by impaired uptake of glucose in response to insulin, is an endogenous protective mechanism which prevents excess uptake of fuel when supply is increased. To test this we fed Sprague-Dawley rats either a high-sucrose diet or regular chow diet. After 5 to 8 weeks systemic insulin sensitivity was impaired in sucrose-fed rats. Hearts were then perfused
ex vivo
in the working mode to measure function and metabolism. Rates of glucose uptake by the heart of sucrose-fed animals were decreased in response to insulin. Cardiac power remained unchanged in hearts perfused with either normal (5mM glucose; 0.4mM oleate; 0.5ng/ml insulin) or high (25mM glucose; 0.8mM oleate; 5ng/ml insulin) concentrations of substrates. However, cardiac power
increased
for the insulin-resistant hearts compared to controls when a hemodynamic stress (afterload raised from 100 to 140cm H
2
O; 1µM epinephrine) was superimposed on nutrient stress. The ratio of glucose-to-oleate oxidation was also markedly increased in these hearts, as was cardiac efficiency. Higher glucose oxidation rates correlated with increased pyruvate dehydrogenase (PDH) activity, and lower uncoupling protein 3 (UCP3) expression. In conclusion, insulin resistance promotes adaptation of the stressed heart by increasing glucose oxidation while limiting excess fuel uptake. Our findings call for a new interpretation of the glucose-fatty acid cycle in the heart.
Abstract P245: Loss of Steroid Receptor Coactivator-2 in the Heart Results in a Return to the Fetal Gene Program
Authors: Reineke Erin L , York Brian , Newgard Christopher B , Taegtmeyer Heinrich , Taffet George E , Entman Mark L , O'Malley Bert W
Published: March 2022

Steroid Receptor Coactivator-2 (SRC-2) is an integral transcriptional mediator of metabolism throughout the body with roles in both fatty acid and carbohydrate metabolism. Based on the critical link between cardiac function and cardiac metabolism, which itself has dynamic interplay between both fatty acid and carbohydrate use, we sought to determine if SRC-2 plays a role in controlling cardiac metabolism. Hearts from SRC-2-/- mice were analyzed by extensive gene expression profiling of major metabolic enzymes in fuel usage pathways as well as metabolomic analysis of major metabolites. Our results indicate that under normal conditions, loss of SRC-2 compromises the ability of the adult heart to metabolize fatty acid as its sole fuel source. We observe a gene expression profile that mimics the fetal heart and a metabolomic profile that suggests a reliance on anaerobic conversion of glucose to lactate for energy. This fetal gene switch is paralleled with changes of sarcomeric gene expression, along with several genes altered in response to stress. Interestingly, this long-term adaptive response is adequate to maintain cardiac function under normal conditions. However, SRC-2-/- mice do show impaired cardiac function as compared to SRC-2+/+ mice under hemodynamic stress from aortic banding. Surprisingly, this is accompanied by the absence of hypertrophy, which may be explained by decreased expression of serum response factor (SRF) and several of the transcription factors with which SRF is known to act. Taken together, loss of SRC-2 results in a cardiac phenotype characterized by a return to the fetal gene program that mimics the state of a stressed and/or aging heart. Further investigation of this mouse model may play an important role in understanding the mechanisms underlying the transcriptional events surrounding cardiac metabolic remodeling.
Abstract P261: Endothelial Differentiation Gene-1 Is Involved in Related Transcriptional Enhancer Factor-1--Induced Endothelial Cell Aggregation
Authors: He Ping , An Xiaojin , Wu Jiaping , Messmer-Blust Angela , Philbrick Melissa , Li Jian
Published: March 2022

We have previously demonstrated that Related Transcriptional Enhancer Factor-1 (RTEF-1) regulated angiogenesis through induction of vascular endothelial growth factor (VEGF) under hypoxic conditions. Recently, we discovered that RTEF-1 accelerated endothelial cell connections and enhanced endothelial cell aggregation. To investigate the mechanism of RTEF-1 in regulated endothelial aggregation, a DNA array analysis was performed in the endothelial cell (HMEC-1) lacking RTEF-1 by its siRNA and resulted in a significant decrease of endothelial differentiation gene -1 (Edg-1). RTEF-1 significantly increase Edg-1 promoter activity (4.10 Fold) and the expression levels of Edg-1 gene are concordant with the RTEF-1 gene in both mRNA and protein levels in cultured endothelial cells as well as in endothelial specific RTEF-1 transgenic mice (VE-Cad-RTEF-1) and RTEF-1 knock out mice (Tie2
Cre
:RTEF-1
floxp
) . In Matrigel tube formation assay, over-expression of Edg-1 gene stimulated endothelial cell aggregation, while siRNA of Edg-1 significantly blocked RTEF-1-driven endothelial cell aggregation. Furthermore, using an Edg-1 receptor agonist Sphingosine-1-phosphate (S1P), the acceleration of the proliferation, migration and tube formation were observed, whereas Edg-1 receptor inhibitor Pertussis Toxin (PTX) could block RTEF-1-induced cell aggregation. Our data demonstrates that Edg-1 is a potential target gene of RTEF-1. It is involved in RTEF-1 regulated endothelial cell angiogenesis, specifically in endothelial cell aggregation.
Abstract P266: Molecular Markers of Endothelial Sensitivity to TNFα
Authors: Mata-Greenwood Eugenia
Published: March 2022

TNF alpha mediated inflammation has been correlated with cardiovascular diseases. Significant interindividual variability to anti-TNF biologicals has been demonstrated in clinical trials. The objective of this study is to unravel the molecular markers of sensitivity to TNF which could be used as predictors of therapeutic efficacy or disease progression. We have isolated and tested a set of 20 HUVECs for their sensitivity to TNF as determined by up-regulation of pro-inflammatory genes such as the Intracellular Adhesion Molecule 1 (ICAM1) and the NADPH oxidase subunit 4 (NOX4). We found that 75% of HUVECs (15/20) responded strongly to TNF compared to the remaining 25% (5/20, p<0.01). Further studies on the TNF signaling pathway indicated that there was a positive correlation between strong response to TNF and higher basal mRNA and protein levels of the mitogen activated protein 3 kinase 1 (MAP3K1 or MEKK1). No further correlations where observed with other signaling components (i.e., TNF receptors, TRAFs, and other MAP3Ks, MAP2Ks or MAPKs). Strong responders also showed 1) increased upregulation of TNFR1 and TRAF5 protein and mRNA levels, 2) higher phosphorylation of JNK and p38 MAPKs and 3) stronger AP1-driven transcription as shown by luciferase assays. Weak responders showed increased TNF-dependent down-regulation of the TNF receptor 1 and of the MAP3K5 (aka Apoptosis-Signaling Kinase 1, ASK1) and TNF-mediated upregulation of the TNF receptor 2 compared to strong responders. Promoter and mRNA stability studies revealed that differences between strong and weak responders in MAP3K1 and MAP3K5 regulation occurs at the transcriptional level. Studies of promoter polymorphisms are undergoing. We conclude that MAP3K1 and MAP3K5 are novel molecular markers of endothelial sensitivity to TNF and future studies on these genes could reveal novel genetic markers for translational research.
Abstract P289: Cardiomyocyte-Specific, but Not Systemic, Rassf1A Deletion Is Protective Against Ischemia-Reperfusion Injury in the Heart
Authors: Del Re Dominic , Zhai Peiyong , Sadoshima Junichi
Published: March 2022

Our previous work demonstrated Rassf1A to be a critical mediator of Mst1 activation, the chief component of the mammalian Hippo pathway, in heart failure. In the setting of ischemia/reperfusion (I/R) injury, Mst1 is robustly activated in the heart; however, its regulation remains unclear. Further, the role of Rassf1A in I/R injury has not been investigated. Using genetically modified mice in which expression of Rassf1A is altered in a cell type-specific manner, we demonstrate that systemic deletion of rassf1a (KO) does not alter infarct size (36±4% vs 33±3%) or cardiac myocyte apoptosis (1.7±0.2% vs 2.1±0.4%) following I/R versus WT. Conversely, mice harboring deletion of rassf1a in cardiac myocytes (CKO) have smaller infarcts (38±3% vs 23±2%, p<0.05) and less apoptosis (2.0±0.2% vs 0.9±0.2%) after I/R. Importantly, attenuation of Mst1 activation in ventricular homogenates was observed in both deletion models, implicating Rassf1A as a positive regulator of Mst1 during I/R. Langendorff global I/R injury yielded similar results - no protection in KO, yet significant protection in CKO hearts versus control mice, suggesting that native cardiac cells are sufficient to mediate this response. A candidate molecule screen found exaggerated TNF-α expression in KO hearts compared to WT (4.8±0.7% vs 1.0±0.3%), whereas TNF-α expression was attenuated in CKO hearts (0.9±0.2% vs 0.3±0.1%). Finally, WT and KO mice were administered TNF-α neutralizing or control IgG and subjected to I/R. KO hearts treated with TNF-α Ab, but not IgG, had reduced infarcts (19±4% vs 34±5%), but no significant reduction in infarct size was observed in WT mice given TNF-α Ab. Taken together these data suggest that TNF-α blockade prevents the deleterious consequences of Rassf1A deletion in non-myocytes while unmasking the protective effect of Rassf1A deletion in cardiac myocytes following I/R. Further, these results demonstrate the importance of non-myocytes in modulating cardiac myocyte survival and I/R injury.
Abstract P311: Differential Changes in Caveolin Levels in Lung and Heart from Ozone-Exposed Adult Healthy Rats
Authors: Sethi Rajat , Ramirez Magdalena , Bandla Ashoka , Manchanda Shubham , Garcia Carlos , Dostal David
Published: March 2022

Acute exposure to ozone (O
3
) has been reported to possess beneficial effects in the injured myocardium. We have demonstrated that chronic O
3
exposure elicits cardiac dysfunction in rats. These results suggest that acute and chronic exposures to O
3
have different effects on myocardial function. On the contrary exposure to acute ozone levels has shown to cause lung injury. Although effects of chronic exposure to ozone on the lung has not been determined, it is reasonable to suggest that ozone mediated toxicity may be tissue specific. It appears that in chronic cardiac injury subsequent to an ischemic insult, lipid raft proteins such as caveolin-1 and caveolin-3 play vital roles in the generation of death and survival signaling in the heart. In lung injury subsequent to acute O
3
exposure, dramatic reductions in caveolin-1 levels were associated with increases in lung TNF-α. Our previous studies showed increased expressions of myocardial TNF-α after chronic O
3
exposure. In the present study we wanted to determine the regulatory role of caveolin-1 and caveolin-3 in hearts and lungs from rats exposed to chronic O
3
levels. Sprague Dawley rats were exposed 8 hrs/day for 28 and 56 days to filtered air or 0.8 ppm O
3
. In order to assess the chronic effects to O
3
, levels of caveolin-1 and caveolin-3 in the lung and heart tissue were measured 24 hrs after termination of the O
3
exposure. Caveolin-1 levels in hearts from the ozone exposed groups were significantly decreased compared to air exposed rats; while caveolin-1 levels in lungs from the same animals were not different from the air exposed group. On the other hand caveolin-3 levels in hearts from the ozone exposed groups increased and decreased after 4 and 8 week of ozone exposure compared to air exposed rats; while caveolin-3 levels in lungs from the same animals were not different from the air exposed group. These novel findings suggest the interesting possibility that caveolin expressions subsequent to chronic ozone exposure may be tissue specific.
Abstract P325: Novel Insights in Transcriptome Remodeling Associated with Heart Failure Revealed by Deep RNA Sequencing
Authors: Gao Chen , Lee Jae-Hyung , Ren Shuxun , Peng Guangdun , MacLellan Robb , Chen Jau-nian , Xiao Grace (Xinshu) , Wang Yibin
Published: March 2022

The complexity of transcriptome and proteome is contributed by alternative splicing of RNA. Altered RNA splicing is also implicated in many human diseases including cancer. However, little knowledge is available about the scope of alternative splicing at whole genome level in heart diseases and even less about the mechanisms underlying the regulation of mRNA splicing in response to pathological injury in heart. In order to investigate the key alternative splicing events associated with cardiac pathogenesis, we used high-throughput RNA-Seq to profile total transcriptome in mouse heart failure induced by pressure-overload. From preliminary data, we identified >1000 novel exons that have not been reported in any published database. Many of the novel exons are detected in genes with potential importance in cardiac gene regulation and signal transduction. In addition, we have also identified a significant number of differentially expressed exons between normal and diseased hearts, supporting the notion that differential RNA splicing is associated with the onset of heart failure. Interestingly, the differential splicing events observed in failing hearts were also detected in neonatal mouse hearts, suggesting that differential RNA splicing is a part of the “fetal” gene expression program in diseased hearts. Furthermore, we have also validated some of these changes in human heart failure samples, supporting the clinic relevance of these findings. In summary, this study suggests that development of heart failure is associated with alternative RNA splicing events at genome level. Further studies on the functional consequences of differential RNA splicing and the underlying regulatory mechanisms in heart will advance our understanding to this important disease.