Redox activation of JNK2α2 mediates thyroid hormone-stimulated proliferation of neonatal murine cardiomyocytes

Mitochondria-generated reactive oxygen species (mROS) are frequently associated with DNA damage and cell cycle arrest, but physiological increases in mROS serve to regulate specific cell functions. T3 is a major regulator of mROS, including hydrogen peroxide (H2O2). Here we show that exogenous thyroid hormone (T3) administration increases cardiomyocyte numbers in neonatal murine hearts. The mechanism involves signaling by mitochondria-generated H2O2 (mH2O2) acting via the redox sensor, peroxiredoxin-1, a thiol peroxidase with high reactivity towards H2O2 that activates c-Jun N-terminal kinase-2α2 (JNK2α2). JNK2α2, a relatively rare member of the JNK family of mitogen-activated protein kinases (MAPK), phosphorylates c-Jun, a component of the activator protein 1 (AP-1) early response transcription factor, resulting in enhanced insulin-like growth factor 1 (IGF-1) expression and activation of proliferative ERK1/2 signaling. This non-canonical mechanism of MAPK activation couples T3 actions on mitochondria to cell cycle activation. Although T3 is regarded as a maturation factor for cardiomyocytes, these studies identify a novel redox pathway that is permissive for T3-mediated cardiomyocyte proliferation—this because of the expression of a pro-proliferative JNK isoform that results in growth factor elaboration and ERK1/2 cell cycle activation.

below) were also diluted in Superblock and incubated for 2 h at 22 °C, or overnight at 4 °C, followed by horseradish peroxidase (HRP)-labeled secondary antibody (1:10,000) for 45 min at 22 °C. The signals were detected using Super Signal West Dura Detection Reagent (Thermo Scientific, 34075) and images captured on a Bio-Rad GelDoc system equipped with CCD camera and ImageLab program (Bio-Rad). Quantification was performed by densitometry using the ImageLab program.

Immunoprecipitation
Cardiomyocytes were collected post-T3 treatment and lysates were generated as detailed in the cell culture protocol (above). To immune-precipitate phosphorylated forms of JNK antigens from the cardiomyocyte lysates, we first incubated primary pan-phosphorylation-specific JNK antibody (1-10 µg) with 50 µl (1.5 mg) immunoglobulin (antibody) binding Protein A Dynabeads (Life technologies, 10001D) for 20 min at room temperature for immobilizing the antibody to the solid Dynabead support. We then placed the tube containing the magnetic Dynabeads on a magnet. The magnetic Dynabeads attached to the inner wall of the tube touching the magnet. This allowed removal of supernatant without disturbing the beads containing the antibody. The supernatant was removed and 200 µl PBS containing 0.02%  added. The tube was detached from the magnet and the PBST solution was gently mixed with the beads. The tube was again placed on magnet and the supernatant containing unbound primary antibody liberated from the Dynabeads by mixing with PBST, was removed. The tube now contained antibody coupled magnetic beads. We then added whole cell protein lysate prepared earlier from T3 treated cardiomyocytes (typically 0.1-1 ml) for 1 to 2 h at room temperature to allow formation of antibody-antigen complexes. At the end of incubation, we washed 3x with 200 µl PBST using the magnet as described earlier. These washings helped purification of only the antibody-antigen bound complex from any unbound non-specific antigens. We then added 20 µl of 2x Laemmli sample buffer (Biorad, 1610737), and after mixing the beads, the sample was heated for 5 min at 95-99 °C and immediately cooled on ice. The tube was then placed on the magnet and the supernatant containing the immunoprecipitated antigen was aspirated and loaded on to an SDS-PAGE gel. Gel running conditions, transfer to PVDF membrane, probing with antibodies and detection were performed as described above.

Non-reducing/reducing SDS-PAGE for detection of multimeric protein complexes
Cardiomyocytes were treated with T3 (10 nmol/L) and collected at 0.5, 1, 2, 3, 4 and 8 h post-T3 treatment. To ensure that the endogenous oxidation and disulfide state of proteins was maintained, cardiomyocytes were immediately washed twice in 500 µl ice-cold PBS supplemented with 10 mg/ml catalase (Sigma-Aldrich, C1345-1G) and 100 mmol/L Nethylmaleimide (NEM, Sigma-Aldrich, E3876-5G). The solution was then aspirated, and the cardiomyocytes were harvested in 250 µl of RIPA buffer supplemented with 10 mg/ml catalase for 20 min on ice. Whole cell cardiomyocyte lysates were generated as detailed in the cell culture protocol above.
For detection of multimeric complexes, we ran the lysates under non-reducing conditions.
We mixed equal volumes of each sample (~10 μl) with equal volume of Native Sample Buffer (Bio-Rad, 161-0738), vortexed briefly and centrifuged at 16,000 x g for 5 min. We collected the supernatant and loaded onto 4-15% Criterion™ TGX™ gel (Bio-Rad, 5671084). The gel was run in pre-chilled 1x running buffer (Tris-Glycine Buffer without SDS; Bio-Rad, 161-0734) and placed the gel tank in iced water. Electrophoresis was performed at 200 volts for 5 min, and then at 150 volts until the loading dye ran through the entire gel. Proteins were then transferred to PVDF membrane, probed with antibody and signal was detected as detailed above in the immuno-blotting protocol. To determine if JNK2/Prx1 multimeric complex are stabilized by disulfide bonds, we added DL-Dithiothreitol (DTT, Sigma-Aldrich, D0632-1G) at a final concentration of 350 mmol/L in the samples diluted in 2x Laemmli sample buffer (Bio-Rad, 161-0737); to reduce disulfide bonds. We heated the samples at 95-99 °C for 5 min and immediately cooled on ice. The samples were then vortexed briefly and centrifuged at 16,000 x g for 5 min.

RT-qPCR
Cardiomyocyte sample tubes containing RNAlater stabilization solution were removed from -80 °C freezer and thawed on ice. The samples were then centrifuged at 21,000 x g for 10 min.
RNAlater supernatant was removed and replaced with 240 µl of lysis binding buffer from the mirVana miRNA Isolation Kit (ThermoFisher, AM1560). RNA was purified according to the manufacturer's guidelines. Purified RNA was reverse transcribed using Transcriptor Reverse Transcriptase (Roche, 03531295001) and random primer (Primer, random p(dN)6, Roche, 11034731001). Quantitative PCR was performed with SYBR Green Supermix (Bio-Rad, 1708882) on a iQ5 Thermal Cycler (Bio-Rad). Primers were synthetized by IDT Technologies; their sequences are presented in Supplementary Table S5.

Cardiomyocyte purification for immunoblotting and immunocytochemistry
For Western blotting and immunocytochemistry, hearts were enzymatically digested, as described above. Before making single cell suspensions, atria were excised, and cardiac cells were disaggregated into single cell suspension. Cardiomyocytes were purified with 3 low speed centrifugations (18 xg for 4 min at room temperature), which caused cardiomyocytes to settle as a pellet. Supernatant fractions, enriched in non-myocytes, were discarded. These cardiomyocyte preparations were > 95% pure (2). Cardiomyocytes were snap frozen in liquid nitrogen and stored at -80 °C for Western blotting. Additionally, cardiomyocytes were fixed with Cytofix (BD Biosciences, 554655) for 5 min and spread on glass slides for immunocytochemistry.
H2O2 measurement: After treating cardiomyocytes with T3 alone or in combination with other reagents tested, the culture medium was collected for H2O2 quantitation using the Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit (Thermo Scientific, A22188). Assays were performed immediately after media collection, according to manufacturer's guidelines.