A small erythropoietin derived non-hematopoietic peptide reduces cardiac inflammation, attenuates age associated declines in heart function and prolongs healthspan

BackgroundAging is associated with increased levels of reactive oxygen species and inflammation that disrupt proteostasis and mitochondrial function and leads to organism-wide frailty later in life. ARA290 (cibinetide), an 11-aa non-hematopoietic peptide sequence within the cardioprotective domain of erythropoietin, mediates tissue protection by reducing inflammation and fibrosis. Age-associated cardiac inflammation is linked to structural and functional changes in the heart, including mitochondrial dysfunction, impaired proteostasis, hypertrophic cardiac remodeling, and contractile dysfunction. Can ARA290 ameliorate these age-associated cardiac changes and the severity of frailty in advanced age?MethodsWe conducted an integrated longitudinal (n = 48) and cross-sectional (n = 144) 15 months randomized controlled trial in which 18-month-old Fischer 344 x Brown Norway rats were randomly assigned to either receive chronic ARA290 treatment or saline. Serial echocardiography, tail blood pressure and body weight were evaluated repeatedly at 4-month intervals. A frailty index was calculated at the final timepoint (33 months of age). Tissues were harvested at 4-month intervals to define inflammatory markers and left ventricular tissue remodeling. Mitochondrial and myocardial cell health was assessed in isolated left ventricular myocytes. Kaplan–Meier survival curves were established. Mixed ANOVA tests and linear mixed regression analysis were employed to determine the effects of age, treatment, and age-treatment interactions.ResultsChronic ARA290 treatment mitigated age-related increases in the cardiac non-myocyte to myocyte ratio, infiltrating leukocytes and monocytes, pro-inflammatory cytokines, total NF-κB, and p-NF-κB. Additionally, ARA290 treatment enhanced cardiomyocyte autophagy flux and reduced cellular accumulation of lipofuscin. The cardiomyocyte mitochondrial permeability transition pore response to oxidant stress was desensitized following chronic ARA290 treatment. Concurrently, ARA290 significantly blunted the age-associated elevation in blood pressure and preserved the LV ejection fraction. Finally, ARA290 preserved body weight and significantly reduced other markers of organism-wide frailty at the end of life.ConclusionAdministration of ARA290 reduces cell and tissue inflammation, mitigates structural and functional changes within the cardiovascular system leading to amelioration of frailty and preserved healthspan

B-MYB Is Essential for Normal Cell Cycle Progression and Chromosomal Stability of Embryonic Stem Cells

Background: The transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1. Methodology/Principal Findings: In the present study, we show that the major cell cycle defect in murine embryonic stem (mES) cells occurs in G2/M. Specifically, knockdown of B-Myb by short-hairpin RNAs results in delayed transit through G2/M, severe mitotic spindle and centrosome defects, and in polyploidy. Moreover, many euploid mES cells that are transiently deficient in B-Myb become aneuploid and can no longer be considered viable. Knockdown of B-Myb in mES cells also decreases Oct4 RNA and protein abundance, while over-expression of B-MYB modestly up-regulates pou5f1 gene expression. The coordinated changes in B-Myb and Oct4 expression are due, at least partly, to the ability of B-Myb to directly modulate pou5f1 gene promoter activity in vitro. Ultimately, the loss of B-Myb and associated loss of Oct4 lead to an increase in early markers of differentiation prior to the activation of caspase-mediated programmed cell death. Conclusions/Significance: Appropriate B-Myb expression is critical to the maintenance of chromosomally stable and pluripotent ES cells, but its absence promotes chromosomal instability that results in either aneuploidy or differentiation-associated cell death

Ascorbic acid promotes cardiomyogenesis through SMAD1 signaling in differentiating mouse embryonic stem cells

<div><p>Numerous groups have documented that Ascorbic Acid (AA) promotes cardiomyocyte differentiation from both mouse and human ESCs and iPSCs. AA is now considered indispensable for the routine production of hPSC-cardiomyocytes (CMs) using defined media; however, the mechanisms involved with the inductive process are poorly understood. Using a genetically modified mouse embryonic stem cell (mESC) line containing a dsRED transgene driven by the cardiac-restricted portion of the <i>ncx1</i> promoter, we show that AA promoted differentiation of mESCs to CMs in a dose- and time-dependent manner. Treatment of mPSCs with AA did not modulate total SMAD content; however, the phosphorylated/active forms of SMAD2 and SMAD1/5/8 were significantly elevated. Co-administration of the SMAD2/3 activator Activin A with AA had no significant effect, but the addition of the nodal co-receptor TDGF1 (Cripto) antagonized AA’s cardiomyogenic-promoting ability. AA could also reverse some of the inhibitory effects on cardiomyogenesis of ALK/SMAD2 inhibition by SB431542, a TGFβ pathway inhibitor. Treatment with BMP2 and AA strongly amplified the positive cardiomyogenic effects of SMAD1/5/8 in a dose-dependent manner. AA could not, however, rescue dorsomorphin-mediated inhibition of ALK/SMAD1 activity. Using an inducible model system, we found that SMAD1, but not SMAD2, was essential for AA to promote the formation of TNNT2⁺-CMs. These data firmly demonstrate that BMP receptor-activated SMADs, preferential to TGFβ receptor-activated SMADs, are necessary to promote AA stimulated cardiomyogenesis. AA-enhanced cardiomyogenesis thus relies on the ability of AA to modulate the ratio of SMAD signaling among the TGFβ-superfamily receptor signaling pathways.</p></div

Proteomic Landscape and Deduced Functions of the Cardiac 14-3-3 Protein Interactome

Rationale: The 14-3-3 protein family is known to interact with many proteins in non-cardiac cell types to regulate multiple signaling pathways, particularly those relating to energy and protein homeostasis; and the 14-3-3 network is a therapeutic target of critical metabolic and proteostatic signaling in cancer and neurological diseases. Although the heart is critically sensitive to nutrient and energy alterations, and multiple signaling pathways coordinate to maintain the cardiac cell homeostasis, neither the structure of cardiac 14-3-3 protein interactome, nor potential functional roles of 14-3-3 protein–protein interactions (PPIs) in heart has been explored. Objective: To establish the comprehensive landscape and characterize the functional role of cardiac 14-3-3 PPIs. Methods and Results: We evaluated both RNA expression and protein abundance of 14-3-3 isoforms in mouse heart, followed by co-immunoprecipitation of 14-3-3 proteins and mass spectrometry in left ventricle. We identified 52 proteins comprising the cardiac 14-3-3 interactome. Multiple bioinformatic analyses indicated that more than half of the proteins bound to 14-3-3 are related to mitochondria; and the deduced functions of the mitochondrial 14-3-3 network are to regulate cardiac ATP production via interactions with mitochondrial inner membrane proteins, especially those in mitochondrial complex I. Binding to ribosomal proteins, 14-3-3 proteins likely coordinate protein synthesis and protein quality control. Localizations of 14-3-3 proteins to mitochondria and ribosome were validated via immunofluorescence assays. The deduced function of cardiac 14-3-3 PPIs is to regulate cardiac metabolic homeostasis and proteostasis. Conclusions: Thus, the cardiac 14-3-3 interactome may be a potential therapeutic target in cardiovascular metabolic and proteostatic disease states, as it already is in cancer therapy

Ascorbic acid promotes cardiomyogenesis in a stage-specific and a dose-dependent manner.

<p><b>A.</b> Flow cytometry analysis of RFP fluorescence in differentiating RFP6 Embryoid Bodies (EBs) at Day 7+2, in AA-treated EBs (Day 2), compared to untreated controls. EBs were plated at Day 7 and fluorescence could be observed as early as Day 7+1. <b>B.</b> RFP6-ESCs (Day 0, n = 3) and RFP6-EBs (Day 2, Day 5, n = 3) were treated with various doses of AA (1 μM, 10 μM, 100 μM) and analyzed for RFP fluorescence at Day 7+2 of differentiation. <b>C.</b> Quantitative PCR analysis of cardiac markers in RFP6-EBs at Day 7+3 of differentiation, after treatment with AA (100 μM) at Day 2 (n = 3). *p<0.05, **p<0.01 and ***p<0.001 vs untreated control.</p

Ascorbic acid-enhanced cardiomyogenesis is SMAD-modulation dependent.

<p>Western Blots of total- and active-SMADs at Day 3 (<b>A</b>) and Day 4 (<b>B</b>) of the differentiation process, after treatments with BMP2 (Day 0), AA (Day 2) and their combination (Day 0 + Day 2). <b>A.</b> Total-SMAD2/3 (n = 6), phospho-SMAD2 (n = 14), total-SMAD1 (n = 6) and phospho-SMAD1/5/8 (n = 16); <b>B.</b> Total-SMAD2/3 and phospho-SMAD2 (n = 8) and total-SMAD1 and phospho-SMAD1/5/8 (n = 10). *p<0.05, **p<0.001 and ***p<0.0001 are relative to untreated control; ^$p<0.05 and ^{$ $$}p<0.001 relative to BMP2 treatment; ^#p<0.05 and ^##p<0.01 relative to AA treatment.</p

Ascorbic acid induction of cardiomyogenesis involves the BMP-signaling cascade.

<p><b>A.</b> Cardiomyogenesis assessment by quantification of RFP⁺-CMs by flow cytometry analysis after treatment with BMP2 (10 ng/mL), added at Day 0, Ascorbic acid, added at Day 2, and their combination (Day 0 + Day 2, n = 5). <b>B.</b> Western Blots of the cardiac markers GATA4 and T following treatment with BMP2 (10 ng/mL, Day 0), AA (Day 2), and their combination (Day 0 + Day 2, n = 6). <b>C.</b> Cardiomyogenesis assessment by flow cytometry quantification of RFP⁺-CMs after dorsomorphin treatment (2 μM), an inhibitor of SMAD1-activation, added at Day 0 or Day 2, alone or in combination with AA (Day 2) (n = 3). <b>D.</b> Western Blots of the cardiac markers GATA4 and T following dorsomorphin inhibition (Day 2, 2 μM, n = 5), alone or in combination with AA (Day 2). *p<0.05, **p<0.01 and ***p<0.001 are relative to untreated control; ^£££p<0.001 relative to BMP2 treatment, ^#p<0.05 relative to AA treatment.</p

Ascorbic acid-mediated cardiogenic-induction is independent of its anti-oxidant and anti-proliferative capacities.

<p><b>A.</b> Cardiomyogenesis (RFP fluorescence) assessed in RFP6-EBs at Day 7+3 after treatment with AA (100 μM), Vitamin E (100 μM) and NAC (1000 μM) (n = 3) performed at Day 2 of differentiation. <b>B.</b> Analysis of DNA content (PI staining) of dissociated EBs at Day 3 of differentiation, 24 hours after treatment with AA (Day 2, 1000 μM, n = 3). The histograms show cell cycle stages G0/G1, S and G2/M. No difference in the percentage of cells could be demonstrated between the treated and untreated controls. *p<0.05 compared to untreated control.</p

Ascorbic acid’s cardiac potential is potentiated by SMAD1.

<p><b>A-B.</b> Western Blot at Day 3 of differentiation of <b>A.</b> total-SMAD1 and phospho-SMAD1/5/8, and <b>B.</b> total-SMAD2/3 and phopsho-SMAD2 in iSMAD1-EBs after induction with doxycycline (Dox, 1 μg/mL, for 24 hours from Day 2 to Day 3), AA induction (Day 2) and their combination, compared to untreated control (iSMAD1-mESCs, n = 6). <b>C.</b> Cardiomyogenesis assessment by flow cytometry quantification of TNNT2⁺-CMs in iSMAD1-EBs at Day 7+3 of differentiation following SMAD1-conditional stimulation by doxycycline (Dox) treatment performed for 24 hours, from Day 2 to Day 3 of the differentiation program, AA induction (Day 2) and their combination, compared to untreated control (iSMAD1-EBss, n = 5). *p<0.05 and **p<0.01 compared to untreated iSMAD1-EBs.</p

Ascorbic acid induction of cardiomyogenesis involves the TGFβ-signaling pathway.

<p><b>A-B.</b> Cardiomyocyte-induction assessed by flow cytometry of RFP⁺-CMs at Day 7+3 of differentiation following treatments with <b>A.</b> Activin A (100 ng/mL) added at Day 2 (n = 12) and <b>B.</b> SB431542 (10 μM), an inhibitor of SMAD2-activation, added at Day 0 (n = 3) or Day 2 (n = 3), performed alone or in combination with AA (Day 2). <b>C.</b> Western Blot of the cardiac markers GATA4 and T assessed at Day 3 of differentiation following treatments with SB431542 (10 μM) and AA, both performed at Day 2 (n = 4). <b>D.</b> Flow cytometry assessment of cardiomyogenesis (RFP⁺-CMs) at Day 7+3 of differentiation following treatments with TDGF1 (100 ng/mL) added at Day 2 (n = 3), alone or in combination with AA (Day 2).*p<0.05, **p<0.01, ***p<0.0001 are compared to untreated control; ^$p<0.01 compared to SB431542 inhibitor; ^££p<0.01 compared to TDGF1 treatment.</p