Remote Ischemic Preconditioning Induces Cardioprotective Autophagy and Signals through the IL-6-Dependent JAK-STAT Pathway

Autophagy is a cellular process by which mammalian cells degrade and assist in recycling damaged organelles and proteins. This study aimed to ascertain the role of autophagy in remote ischemic preconditioning (RIPC)-induced cardioprotection. Sprague Dawley rats were subjected to RIPC at the hindlimb followed by a 30-min transient blockade of the left coronary artery to simulate ischemia reperfusion (I/R) injury. Hindlimb muscle and the heart were excised 24 h post reperfusion. RIPC prior to I/R upregulated autophagy in the rat heart at 24 h post reperfusion. In vitro, autophagy inhibition or stimulation prior to RIPC, respectively, either ameliorated or stimulated the cardioprotective effect, measured as improved cell viability to mimic the preconditioning effect. Recombinant interleukin-6 (IL-6) treatment prior to I/R increased in vitro autophagy in a dose-dependent manner, activating the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathway without affecting the other kinase pathways, such as p38 mitogen-activated protein kinases (MAPK), and glycogen synthase kinase 3 Beta (GSK-3β) pathways. Prior to I/R, in vitro inhibition of the JAK-STAT pathway reduced autophagy upregulation despite recombinant IL-6 pre-treatment. Autophagy is an essential component of RIPC-induced cardioprotection that may upregulate autophagy through an IL-6/JAK-STAT-dependent mechanism, thus identifying a potentially new therapeutic option for the treatment of ischemic heart disease.


Blood collection
Immediately post-RIPC, abdominal incision was made on the anesthetized rats and renal artery visualized, and 3−4 mL of blood was withdrawn with a 22-gauge hypodermic needle and quickly transferred into a Lithium-Heparin Blood collection tube (BD Biosciences, New Jersey, USA) and kept on ice till centrifugation. Blood was centrifuged at 2500 rpm for 15 min. Blood plasma was collected in a 1.5-mL Eppendorf tube and stored at −80°C.

In vitro apoptosis study
After the experiments, H9c2 cells were washed with cold D-PBS twice. Followed by trypsinization, cells were centrifuged at 1000 rpm for 5 min. Cell pellet was suspended in 1x binding buffer at a concentration of 1 x 10^6 cells/mL. In total, 100L of the solution was transferred to FACS tubes. Then, 5L of Annexin V FITC and 5L of propidium iodide (PI) was added to the tube and incubated for 15 min at room temperature in the dark. Prior to running the sample in the flow cytometry machine, 400L of 1x binding buffer was added to each tube and the apoptotic cells were determined by analyzing 10,000 gated cells using a BD FACS flow cytometer.

Mitochondrial membrane potential loss study
After the experiments, H9c2 cells were washed with cold D-PBS twice. Followed by trypsinization, cells were centrifuged at 1000 rpm for 5 min. Cell pellet was suspended in 1mL of D-PBS at a concentration of 1 x 10^6 cells/mL. Briefly, 5L of 2M stock of DiLC5 dye was added to the cells and incubated at room temperature for 15 min in the dark. The fluorescence intensity of the cells was analyzed by gating 10,000 cells using a BD FACS flow cytometer.

Cell viability assay
Cell viability was measured using a Live/Dead Cell Imaging Kit (#R37601, Thermofisher Scientific, Massachusetts, USA). Cells were seeded in a 6-well plate, and after experiments, wells were washed 2 times with D-PBS. Live Green vial from the kit was thawed and transferred to the Dead Red vial to prepare 2x stock. Equal volumes of 2x stock and live cell imaging solution (140mM NaCl, 2.5mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 20mM HEPES, pH 7.4) were incubated at room temperature for 15 min. Fluorescence images were taken using GFP and Texas Red filter in an EVOS FL Auto Cell Imaging System (Thermofisher Scientific, Massachusetts, USA).

Preparation of sample for RNA extraction
After exposure to the experimental condition, cells were washed with ice-cold D-PBS, and trypsinized cells (5x10 6 to 1x10 7 cells) were centrifuged at 14,000 rpm. Cell pellet was resuspended in 500L of lysis solution/ 2-ME mixture, and total RNA extracted using the Gen elute Mammalian Total RNA Mini Prep Kit (Sigma-Aldrich, St Louis, Missouri, USA).
Tissue sample was excised and washed with ice-cold D-PBS and "snap-frozen" in dry ice. In total, 500L of lysis solution/2-ME mixture was added to the frozen tissue and homogenized in a flat bottom DNAase, RNAse-free cell culture tube on ice with TissueRuptor (Qiagen, USA). Total RNA was isolated using the Gen elute Mammalian Total RNA Mini Prep kit.

Total RNA concentration measurement
The concentration of total RNA was measured from 2L of the extracted RNA sample using a NanoDrop 1000 Spectrophotometer (Thermofisher Scientific, USA).

cDNA synthesis
RNA was reverse transcribed to 1g cDNA using the Tetro cDNA Synthesis Kit (Bioline, London, UK) in accordance with the manufacturer's instructions. cDNA was stored in −20°C.

Real-Time PCR
RT-PCR mixture prepared using the SensiFast SYBR HI ROX Kit (Bioline, London, UK) in accordance with the manufacturer's instructions. Initially, serial dilution of cDNA sample was done, and RT-PCR was run to generate a standard curve to estimate the efficiency of the primers. RT-PCR was performed in an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA). Reactions were performed in at least triplicate and were analyzed by relative quantitation using RQ Manager software, version 1.2 (Applied Biosystems).

ELISA
Enzyme-linked immunosorbent assay (ELISA) kits were used to determine the concentration of IL-6 (Rat IL-6 Quantikine ELISA Kit, R&D Systems, Minneapolis, USA) protein. For circulating protein levels, blood from rats was collected in a Lithium-Heparin tube and spun at 2500 rpm for 5 min and plasma was collected. For the detection of plasma IL-6, 50 L of rat plasma was used and protein estimated as per the manufacturer's instructions.

Autophagy promotion and inhibition
Recombinant IL-6 was added to hypoxic media, and H9c2 cells were exposed to recombinant IL-6-containing hypoxic media during 30 min of hypoxia. In order to optimize the rapamycin concentration to induce autophagy, we treated overnight serum-starved H9c2 cells with different concentrations of rapamycin for 4 h in serum-free DMEM. 3-MA blocks autophagic sequestration and autophagosome formation by inhibiting class III phosphatidylinositol 3-kinase (PI3K) [1,2]. Thus, 3-MA downregulates autophagic activity at the early stage and is widely used to understand the role of autophagy [3][4][5]. In previous studies, pre-treatment with 10 mM 3-MA for 2 h has been shown to effectively block autophagic activity [6]. Hence, H9c2 cells were treated with 10nM 3-MA for 2 h in serum-free DMEM. Cells were pre-treated with 200 μM Tyrphostin AG 490 for 1 h prior to exposing the cells to H/R to inhibit the JAK-STAT pathway as previously described [7].