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UCP2 modulates cardiomyocyte cell cycle activity, acetyl-CoA, and histone acetylation in response to moderate hypoxia

Developmental cardiac tissue is regenerative while operating under low oxygen. After birth, ambient oxygen is associated with cardiomyocyte cell cycle exit and regeneration. Likewise, cardiac metabolism undergoes a shift with cardiac maturation. Whether there are common regulators of cardiomyocyte cell cycle linking metabolism to oxygen tension remains unknown. The objective of the study is to determine whether mitochondrial UCP2 is a metabolic oxygen sensor regulating cardiomyocyte cell cycle. Neonatal rat ventricular myocytes (NRVMs) under moderate hypoxia showed increased cell cycle activity and UCP2 expression. NRVMs exhibited a metabolic shift toward glycolysis, reducing citrate synthase, mtDNA, mitochondrial membrane potential (ΔΨm), and DNA damage/oxidative stress, while loss of UCP2 reversed this phenotype. Next, WT and mice from a global UCP2-KO mouse line (UCP2KO) kept under hypoxia for 4 weeks showed significant decline in cardiac function that was more pronounced in UCP2KO animals. Cardiomyocyte cell cycle activity was reduced, while fibrosis and DNA damage was significantly increased in UCP2KO animals compared with WT under hypoxia. Mechanistically, UCP2 increased acetyl-CoA levels and histone acetylation, and it altered chromatin modifiers linking metabolism to cardiomyocyte cell cycle under hypoxia. Here, we show a potentially novel role for mitochondrial UCP2 as an oxygen sensor regulating cardiomyocyte cell cycle activity, acetyl-CoA levels, and histone acetylation in response to moderate hypoxia.


Cell Isolation and culture
Neonatal rat ventricular myocytes (NRVMs) were isolated from 1-2 day old rat pups as previously described (1). NRVMs were plated at a density of 2.5 X 10 5 cells/well in a 2-well chamber slide, 5.0 X 10 5 cells/6-cm dish, and 1.0 X 10 6 cells/10-cm dish. The cells were maintained overnight in F-10 Nutrient Mix (Gibco), supplemented with 10% Horse Serum, Fetal Bovine Serum, and antibiotics (100 units/mL penicillin and 100 mg/mL streptomycin). Adult feline ventricular myocytes were isolated as described previously (2) and cultured in M199 medium supplemented with penicillin, streptomycin and gentamicin. Cells were incubated in a 37°C 5% CO2 and 20% O2.

Immunoblots
Immunoblot analysis was performed as previously described (1,3,4). In short, protein lysates were loaded onto a 4 -20% Mini-PROTEAN TGX Gel (Biorad) for electrophoresis. Separated proteins were then transferred onto a nitrocellulose membrane, washed with 1X PBS, and blocked with PBS-based Licor Buffer for 1 hour at 4°C. This was followed by primary antibody incubation overnight. Respective secondary antibody cocktails were added for 90 minutes and fluorescence signal was detected and quantified using Odyssey CLx Imager (Licor) and ImageJ.
Immunostaining of NRVMs was performed on cells grown on permanox or glass chamber slides.
Cells were fixed by 4% paraformaldehyde (PFA), permeabilized in PBS supplemented by 0.25% Triton-X for 10 min and blocked in PBS supplemented with 10% horse serum for 1 hr. Primary antibodies diluted were applied overnight at 4°C after blocking in PBS with 10% horse serum. The next day, cells were washed with PBS and incubated for 1 h at room temperature with secondary antibodies diluted in blocking solution and then stained for DAPI (1:10.000) for 10 minutes.
Vectashield (Vector Labs, CA, USA) mounting media was used to prepare the slides.
Paraffin heart sections were deparaffinized in xylene and rehydrated through graded alcohols to distilled water. Antigen retrieval was achieved by boiling the slides in 10 mmol/L citrate pH 6.0 for 12-15 min. Slides were washed several times with distilled water and once with TN buffer (100 mmol/L Tris, 150 mmol/L NaCl). Slides were then washed in TN buffer and blocked in TNB buffer (TSATM kit from Perkin-Elmer) at room temperature for at least 1 hr. Primary antibodies were applied overnight at 4°C in TNB buffer. The next day, samples are washed in TN buffer and incubated with secondary antibodies at room temperature in the dark for 1 hr. Slides are washed in TN buffer and coverslipped using Vectashield in the presence of DNA staining. List of primary and secondary antibodies is reported in Table S2.
EdU labelling and apoptotic cells were assessed by using Click-iT EdU Alexa Fluor 488 Imaging Kit (Thermo Fisher Scientific) and Click-iT™ Plus TUNEL Assay (Thermo Fisher Scientific), respectively, as described by the manufacturer. Fibrotic area was assessed by Masson's trichrome staining (Sigma-Aldrich) following protocol described by the manufacturer and the images were analyzed using ImageJ software.

Citrate Synthase Activity
Citrate synthase activity was measured using citrate synthase kit (Biovision) according to manufacturer's protocol. Briefly, NRVMs were grown to confluency followed by lyses using icecold CS Assay Buffer. Lysate was kept on ice for 10 minutes then centrifuged at 10,000 RPM for 5 minutes. Once supernatant was collected, samples were added to a 96-well flat bottom plate.
Total volume of 50µL was achieved by adding appropriate amount of CS Assay Buffer. Diluted CS Positive Control was added in specific wells and adjusted to total 50µL with CS Assay Buffer.
Standard curve preparation involved diluting GSH Standard and adding to wells. Reaction mix consisting of CS Assay Buffer, CS Developer, and CS Substrate Mix was then added to each well containing either sample, Positive Control, or Standards. Absorbance (OD 412 nm) was measured in kinetic mode at 25°C for 20-40 minutes. Calculation of citrate synthase activity was done using manufacturer's equation.

Mitochondrial DNA
For assessment of Mitochondrial DNA content, cells were centrifuged into a pellet, supernatant was aspirated and DNA lysis buffer (0.5% SDS, 0.1M NaCl, 0.05M Tris (pH 8.0), 3mM EDTA, and ddH20) was added to cells. Proteinase K is added at 100µg/mL to digest any contaminating proteins. Samples were incubated at 60°C overnight and then mixed with 8M Potassium Acetate.
Chloroform was added to sample followed by centrifugation for 5 minutes at 9500rpm. Aqueous phase was isolated and 100% EtOH was added and stored in -20°C freezer for 15 minutes. After centrifugation for 5 minutes at 13000 rpm, supernatant was removed, and pellet was washed with 75% EtOH. Samples were centrifuged for 2 minutes at 1300 rpm and supernatant was removed.
Once air-dried, samples were resuspended in 100 µL ddH2O. DNA samples were normalized to 100ng/µl. qPCR was performed with primers for β-globin representing nuclear genome and COXII for mitochondrial genome. Mitochondrial genome content was measured after normalizing samples.

Echocardiography
Transthoracic two-dimensional echocardiography was performed at baseline and after 4 weeks and percentage ejection fraction are calculated as described previously (1,5). Speckle-tracking based strain analysis on echocardiography B-mode loops with a frame rate ≥200 frame/second and consisted of 300 frames was also performed for all animal groups. All images were analyzed using the Vevo Strain Software (Vevo LAB 1.7.1) for strain, which evaluates changes in length relative to the initial length (strain = final length [L]/initial length [L0]), calculated either in the radial axis (from the center of the ventricle cavity outward) or longitudinal axis (from the apex to the base). The strain rate (SR), which is the rate of change of this deformation over time (SR = strain/time), was also measured. Global and regional (six segments: basal, mid, and apical anterior; basal, mid, and apical posterior) LV endocardial longitudinal and radial strain (peak strain %), as well as SR were evaluated. Regional LV endocardial longitudinal and radial strain/SR were reported as apical versus mid versus basal segments and as anterior (average of basal, mid, and apical) versus posterior (average of basal, mid, and apical).

Acetyl Co-A measurement
Acetyl-CoA were quantified by stable isotope dilution liquid chromatography-high resolution mass spectrometry, as previously described (6,7). Cell pellets were spiked with a 13C315N1-internal standard mixture biosynthetically prepared as previously published and sonicated for 12 cycles of 0.5 sec. pulses in 10% (w/v) trichloroacetic acid (Sigma Aldrich) in water. Protein was pelleted by centrifugation at 17,000rcf for 10 min. at 4ºC. The cleared supernatant was purified by solidphase extraction using Oasis HLB 1cc (30 mg) SPE columns (Waters). Columns were washed with 1 mL methanol, equilibrated with 1 mL water, loaded with sample, desalted with 1 mL water, and eluted with 1 mL methanol containing 25 mM ammonium acetate. The purified extracts were evaporated to dryness under nitrogen and resuspended in 55 μl 5% (w/v) 5-sulfosalicylic acid (SSA) in optima HPLC grade water. Acetyl-CoA was measured by liquid chromatography-high resolution mass spectrometry. Briefly, 5 μl of sample in 5% SSA were analyzed by injection into an Ultimate 3000 HPLC coupled to a Q Exactive Plus (Thermo Scientific) mass spectrometer in positive ESI mode using the settings described previously. Calibration curves were prepared using commercially available standards from Sigma Aldrich and processed identically as the samples. Data were integrated using Tracefinder v4.1 (Thermo Scientific) software, and additional statistical analysis conducted by Prism v7.05 (GraphPad). Acetyl-CoA values were normalized to cell number and reported as pmol/105 cells.

RT2 Profiler Array
Total RNA was isolated from cultured NRVMs using RNeasy Mini Kit (Qiagen) with DNase I (Qiagen) treatment. Total RNA was then reverse transcribed using the First Strand Synthesis Kit (Qiagen) and subsequently loaded on to Epigenetic Chromatin Modification Enzymes RT 2 profiler array according to manufacturer's instructions (Qiagen). Qiagen's online web analysis tool was utilized to produce comparative heat map, and volcano plot. Fold change was calculated by determining the ratio of mRNA levels to control values using the ΔCt method (2 −ΔΔCt ). All data were normalized to an average of four housekeeping genes Actb, Gapdh, B2m, Rplp1, and Hprt.