Mybl2 rejuvenates heart explant-derived cells from aged donors after myocardial infarction.

While cell therapy is emerging as a promising option for patients with ischemic cardiomyopathy (ICM), the influence of advanced donor age and a history of ischemic injury on the reparative performance of these cells are not well defined. As such, intrinsic changes that result from advanced donor age and ischemia are explored in hopes of identifying a molecular candidate capable of restoring the lost reparative potency of heart explant-derived cells (EDCs) used in cell therapy. EDCs were cultured from myocardial biopsies obtained from young or old mice 4 weeks after randomization to experimental myocardial infarction or no intervention. Advanced donor age reduces cell yield while increasing cell senescence and the secretion of senescence-associated cytokines. A history of ischemic injury magnifies these effects as cells are more senescent and have lower antioxidant reserves. Consistent with these effects, intramyocardial injection of EDCs from aged ischemic donors provided less cell-mediated cardiac repair. A transcriptome comparison of ICM EDCs shows aging modifies many of the pathways responsible for effective cell cycle control and DNA damage/repair. Over-expression of the barely explored antisenescent transcription factor, Mybl2, in EDCs from aged ICM donors reduces cell senescence while conferring salutary effects on antioxidant activity and paracrine production. In vivo, we observed an increase in cell retention and vasculogenesis after treatment with Mybl2-over-expressing EDCs which improved heart function in infarcted recipient hearts. In conclusion, Mybl2 over-expression rejuvenates senescent EDCs sourced from aged ICM donors to confer cell-mediated effects comparable to cells from young nonischemic donors.

Direct and Indirect Suppression of Scn5a Gene Expression Mediates Cardiac Na+ Channel Inhibition by Wnt Signalling.

BACKGROUND: Myocardial infarction and heart failure are associated with reduced voltage-gated Na+ current (INa) that promotes arrhythmias and sudden deaths. We have previously shown that the Wnt/ß-catenin signalling (Wnt signalling), which is active in heart disease, reduces cardiac INa, suggesting that Wnt signalling may be a potential therapeutic target. However, because Wnt signalling is required for the homeostasis of many noncardiac tissues, administration of Wnt inhibitors to heart patients would cause significant side effects. The present study aims to elucidate the molecular mechanisms of cardiac INa inhibition by Wnt, which would identify cardiac-specific therapeutic targets. METHODS: Wnt signalling was activated in neonatal rat ventricular myocytes by Wnt3a protein. Adenovirus expressing Wnt3a was injected into the adult rat ventricle. CRISPR/Cas9 and chromatin immunoprecipitation were used for mechanistic studies. RESULTS: Wnt signalling activation in neonatal rat ventricular myocytes reduced Nav1.5 protein and Scn5a mRNA, but increased Tbx3, a known suppressor of Scn5a. Chromatin immunoprecipitation showed that Wnt signalling inhibits Scn5a expression through downstream mediator (TCF4) binding to both Tbx3 and Scn5a promoters. Overexpression or knockdown of Tbx3 directly modified Nav1.5 and INa, whereas CRISPR/Cas9-induced mutations at TCF4 binding sites within the Scn5a promoter attenuated Wnt inhibition of Scn5a and Nav1.5. In adult rat hearts, adenovirus expressing Wnt3a reduced Nav1.5, increased QRS duration in electrocardiogram, and increased the susceptibility to ventricular tachycardia. CONCLUSIONS: Wnt signalling inhibits the Na+ channel by direct and indirect (via Tbx3) suppression of Scn5a transcription. Strategies to block TCF4 binding to the Tbx3 and Scn5a promoters would represent novel strategies for cardiac-specific inhibition of the Wnt pathway to rescue INa and prevent sudden cardiac deaths.

Development of an inflammation imaging tracer, 111In-DOTA-DAPTA, targeting chemokine receptor CCR5 and preliminary evaluation in an ApoE-/- atherosclerosis mouse model.

BACKGROUND: Chemokine receptor 5 (CCR5) plays an important role in atherosclerosis. Our objective was to develop a SPECT tracer targeting CCR5 for imaging plaque inflammation by radiolabeling D-Ala-peptide T-amide (DAPTA), a CCR5 antagonist, with 111In. METHODS: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated DAPTA (DOTA-DAPTA) was labeled with 111In. Cell uptake studies were conducted in U87-CD4-CCR5 and U87-MG cells. Biodistribution was determined in C57BL/6 mice. Autoradiography, en face and Oil Red O (ORO) imaging studies were performed in ApoE-/- mice. RESULTS: DOTA-DAPTA was radiolabeled with 111In with high radiochemical purity (> 98%) and specific activity (70 MBq·nmol). 111In-DOTA-DAPTA exhibited fast blood and renal clearance and high spleen uptake. The U87-CD4-CCR5 cells had significantly higher uptake in comparison to the U87-MG cells. The cell uptake was reduced by three times with DAPTA, indicating the receptor specificity of the uptake. Autoradiographic images showed significantly higher lesion uptake of 111In-DOTA-DAPTA in ApoE-/- mice than that in C57BL/6 mice. The tracer uptake in 4 month old ApoE-/- high fat diet (HFD) mice with blocking agent was twofold lower than the same mice without the blocking agent, demonstrating the specificity of the tracer for the CCR5 receptor. CONCLUSION: 111In-DOTA-DAPTA, specifically targeting chemokine receptor CCR5, is a potential SPECT agent for imaging inflammation in atherosclerosis.

Evaluation of Apoptosis with 99mTc-rhAnnexin V-128 and Inflammation with 18F-FDG in a Low-Dose Irradiation Model of Atherosclerosis in Apolipoprotein E-Deficient Mice.

Low-dose radiation in apolipoprotein E-deficient (ApoE-/-) mice has a protective effect with less subsequent atherosclerosis. Inflammation and apoptosis play major roles in the development of atherosclerosis. We evaluated the temporal pattern of the development of histologic atherosclerosis, inflammation with 18F-FDG, and apoptosis with 99mTc-rhAnnexin V-128 at 3 time points. METHODS: ApoE-/- mice were fed a high-fat diet, exposed to low-dose 60Co γ-radiation of 25 mGy at 2 mo of age, and evaluated within 1 wk (2-mo group), 1 mo (3-mo group), and 2 mo (4-mo group) from the time of radiation. Mice were divided into 3 subgroups and each received 18F-FDG, 99mTc-rhAnnexin V-128, or no radiotracer for autoradiography. Mice underwent euthanasia and aortic root dissection. The extent of atherosclerosis was determined by en face and Oil red O imaging. Aortic arch inflammation (18F-FDG) and apoptosis (99mTc-rhAnnexin V-128) were determined with digital autoradiography. Aortic sinus sections were stained with Sudan IV for assessment of lesion area and stage, antiCD68 antibody for inflammation and anti-cleaved-caspase 3 antibody for apoptosis. RESULTS: The extent of aortic atherosclerosis increased from 2 to 3 mo and from 3 to 4 mo. Inflammation (CD68) decreased and apoptosis (anti-cleaved-caspase 3 antibody) increased in aortic sinus slices measured as percentage of lesion by 4 mo. With increasing lesion stage, lesion inflammation decreased and lesion apoptosis increased. Aortic arch inflammation (18F-FDG uptake) did not differ over time and did not correlate with average lesion stage. However, aortic arch apoptosis (99mTc-rhAnnexin V-128) increased significantly by 4 mo and correlated with average lesion stage. There were no differences between the treatment subgroups (18F-FDG, 99mTc-rhAnnexin V-128, or no radiotracer). CONCLUSION: The temporal pattern of development of inflammation and apoptosis differ during the development of atherosclerosis in ApoE-/- mice treated with low-dose radiation. Advanced lesions are characterized by increased apoptosis and either less or similar amounts of inflammation, shown on immunohistochemistry and autoradiography. Treatment with radiotracers had no significant effects on extent of atherosclerosis, inflammation, or apoptosis.

Glucocorticoid receptor signaling is essential for mesoderm formation and muscle development in zebrafish.

Glucocorticoid receptor (GR) signaling is thought to play a key role in embryogenesis, but its specific developmental effects remain unclear. Cortisol is the primary ligand for GR activation in teleosts, and in zebrafish (Danio rerio), the prehatch embryo content of this steroid is of maternal origin. Using early zebrafish developmental stages, we tested the hypothesis that GR signaling is critical for embryo growth and hatching. In zebrafish, maternal GR mRNA is degraded quickly, followed by zygotic synthesis of the receptor. GR protein is widely expressed throughout early development, and we were able to knockdown this protein using morpholino oligonucleotides. This led to a more than 70% reduction in mRNA abundance of matrix metalloproteinase-13 (mmp13), a glucocorticoid-responsive gene. The GR morphants displayed delayed somitogenesis, defects in somite and tail morphogenesis, reduced embryo size, and rarely survived after hatch. This correlated with altered expression of myogenic markers, including myogenin, myostatin, and muscle-specific myosin heavy chain and troponin genes. A key finding was a 70-90% reduction in the mRNA abundance of bone morphogenetic proteins (BMP), including bmp2a, bmp2b, and bmp4 in GR morphants. Bioinformatics analysis confirmed multiple putative glucocorticoid response elements upstream of these BMP genes. GR morphants displayed reduced expression of BMP-modulated genes, including eve1 and pax3. Zebrafish GR mRNA injection rescued the GR morphant phenotype and reversed the disrupted expression of BMP and myogenic genes. Our results for the first time indicate that GR signaling is essential for zebrafish muscle development, and we hypothesize a role for BMP morphogens in this process.