Effects of Interval Training Effects on Cardiac Fibrosis

Overview

The study aimed to highlight the effect of high-intensity interval training (HIIT) on cardiac fibrosis in cardiac patients. From 2009-2018, cardiac patients with measurements of VO2peak, b-type natriuretic peptide, quality of life questionnaire, cardiovascular magnetic resonance imaging with late gadolinium enhancement (CMR-LGE), and preserved serum before and after 36 times of HIIT were enrolled. The human cardiac fibroblast (CF) isolated from human adult ventricle is treated with sera before and after HIIT. Measurements of cell migration as well as cell proliferation and global cell protein profiles before and after HIIT will be performed.

From January 1, 2009 to December 31, 2018, cardiac patients with stable clinical status for greater than 4 weeks have completed 36 times of HIIT. Subjects with clinical assessments (listed below) before and after HIIT will be included. \*Baseline information, including age, gender, body mass index, duration disease duration, co-morbidities and medication history were reviewed. Subjects with echocardiographic examination, short form-36 health survey (SF-36) for qualities of life, graded cardiopulmonary exercise test (CPET), cardiac magnetic resonance imaging with late gadolinium enhancement (CMR-LGE), blood chemistry tests (hematocrit, brain natriuretic peptide, high-sensitivity C-reactive protein, creatinine), and preserved sera before and after HIIT.\* Cell behaviors Human cardiac fibroblast (CF) isolated from human adult ventricle (HCF-av cell, ScienCell Research Laboratories, Carlsbad, CA, USA) are cultured in medium containing 10 % FBS, 1% fibroblast growth supplement, and 1% penicillin/streptomycin. Cells (1x10\^5) were seeded in dishes of 10-cm diameter and were cultured in the above medium overnight. We used 10% of the patient serum in substitution for the 10% fetal bovine serum (FBS) to treat human cardiac fibroblast (CF) isolated from human adult ventricle (HCF-av cell, ScienCell Research Laboratories, Carlsbad, CA, USA), and serum effects on CF were evaluated. Cell Culture Human cardiac fibroblasts (CFs) isolated from human adult ventricle (HCF-av cell, ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in medium containing 10 % FBS, 1% fibroblast growth supplement, and 1% penicillin/streptomycin. Cells (1x10\^5) were seeded in dishes of 10-cm diameter and were cultured in the above medium overnight. Cell migration assay The cell migration speed in the serum obtained from cardiac patients pre- and post-HIIT was determined as described previously. In brief, HCF-av cells of 3000 were plated on each 3.5-cm Petri dishes with polyacrylamide substrates and were harvest in media containing 10% of cardiac patient sera before and after HIIT. Phase contrast images were recorded using a cooled charge-coupled device camera (Photometrics, Tucson, AZ), attached to an Eclipse Ti-E inverted microscope system (Nikon Instruments Inc., Melville, NY) equipped with a 20X, numerical aperture 0.75 Achromat phase objective lens and a INU series stage top incubator (Tokai HIT Co., Ltd., Shizuoka-ken, Japan). The position of the cell was determined every 10 min for a period of 120-180 mins, based on the center of the nucleus. Migration speed was calculated based on the persistent random walk equation. Cell proliferation assay HCF-av cells of 7.5x10\^3 were plated in each well (0.95 cm\^2 growth area) of a 48-well cell culture plate (Sigma-Aldrich, St. Louis, MO) with ordinary medium for 8 hours. They were then cultured in a starvation medium containing 1.5% FBS overnight. The prepared cells were stained with Hoechst 33342 (Thermo Fisher Scientific Inc., Waltham, MA) for 15 mins and were then washed twice in phosphate buffered saline. They were separately treated with 10% FBS (10 wells), and 10% patient serum obtained from our subjects before (24 wells) and after HIIT (24 wells). We used IN Cell Analyzer 1000 cellular imaging and analysis system (GE Healthcare Bio-Science Corp., Piscataway, NJ) to count cell numbers at 0, 24 hours, and 48 hours after harvested with the three different culture media. Proteomic analysis Protein concentrations of different treatment groups were determined by Bicinchoninic acid assay (ThermoFisher Scientific Inc.). Protein mixtures were separated by electrophoresis on 12.5% SDS-PAGE gel followed by in-gel enzyme digestion according to the following procedure. After thermal denaturation at 95 oC for 5 min, protein samples were reduced through the addition of dithiothreitol (DTT) to a final concentration of 10 mM and incubated at 50 oC for 30 min. Alkylation was performed by adding iodoacetamide (IAA) to a final concentration of 20 mM prior to incubation at room temperature for 30 min in the dark. A second aliquot of DTT was then added to quench unreacted IAA. For trypsin digestion, trypsin was added (1:50, w/w) and the reaction mixture was incubated at 37 oC for 12 h. The trypsin digestion was quenched through the addition of a 10 microL formic acid (10%). Digested peptides were dried by speedvac for mass analysis. The tryptic peptides were analyzed on a LTQ-FT (linear quadrupole ion trap-Fourier transform ion cyclotron resonance) hybrid mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA) equipped with a nano-electrospray ion source (New Objective, Inc., Woburn, MA) in positive ion mode. The liquid chromatography system was the Agilent 1100 Series HPLC (Agilent Technologies, Palo Alto, CA) with the Famos autosampler (LC Packings, San Francisco, CA). Peptide solution was injected onto a self-packed precolumn (150 microm I.D. x 20 mm, 5 microm, 200 Å) and the chromatographic separation was sequentially performed on a self-packed reversed phase C18 nano-column (75 microm I.D. x 300 mm, 5 microm, 100 Å) by using 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in 80% acetonitrile (mobile phase B). A linear gradient from 5 to 40% mobile phase B for 40 min at a flow rate of 300 nL/min was applied. Electrospray voltage was applied at 2.0 kV and capillary temperature was set at 200 oC. A scan cycle was initiated with a full-scan survey MS spectrum (m/z 300 - 2000) performed on the FT-ICR mass spectrometer with resolution of 100,000 at 400 Da. Ten most abundant ions detected in this scan were subjected to a MS/MS experiment performed in the linear quadrupole ion trap (LTQ) mass spectrometer. Ion accumulation (Auto Gain Control target number) and maximal ion accumulation time for full-scan and MS/MS were set at 1 x 106 ions, 1000 ms and 5 x 104 ions, 200 ms. Ions were fragmented by use of CID (collision induced dissociation) with the normalized collision energy was set to 35 %, activation Q was 0.3 and activation time was 30 ms. All experiment RAW files were subjected to MaxQuant (1.5.3.30) for the label-free protein quantitation. The variable post-translational modifications of search parameters in MaxQuant were assigned to include the oxidation of methionine, and the phosphorylation of serine/threonine/tyrosine. The carbamidomethylation of cysteine was assigned as the fixed modification. The enzyme for digestion was assigned to be trypsin with the miss cleavage number two. Homo Sapiens protein sequences from SwissProt 2018\_06 were used for MS/MS search. Protein quantitation results were obtained by the LFQ intensity from MaxQuant. Statistical analysis Wilcoxon matched-pairs signed-ranks test was used to compare exercise capacity parameters, CMR-LGE results, blood chemistry data, and SF-36 scores before and after HIIT in each individual. Student t test was used to assess protein level changes before and after HIIT.