Wednesday, June 7, 2023
BestWooCommerceThemeBuilttoBoostSales-728x90

Insulin signaling is critical for sinoatrial node maintenance and function – Experimental & Molecular Medicine


Mice

Homozygous IGF-1Rfl/fl and IRfl/fl mice13,14,15 were bred with Hcn4-CreErt2 or Hcn4- CreErt2;ROSA26TomRed mice16 to generate conduction cell–specific KO mice, including IGF-1Rfl/fl;Hcn4-CreErt2;ROSA26TomRed (CSIGF1RKO), IRfl/fl;CreErt2;ROSA26TomRed (CSIRKO), and IGF-1Rfl/fl;IRfl/fl; CreErt2;ROSA26TomRed (CSDIRKO) mice. To delete the locus of X-over P1 (loxP) site by activating CreErt2 in mice, 40 mg/kg tamoxifen was administered by intraperitoneal injection to the mice for 4 days. Non-floxed CreErt2;ROSA26TomRed mice were also injected with tamoxifen and served as the wild-type (WT) group. Animals were fed standard chow and autoclaved water ad libitum and housed in temperature-controlled, pathogen-free facilities with a 12/12 h light/dark cycle. All animals were maintained on a C57BL/6J background, and male mice were used for all experiments. All animal experiments were conducted according to the guidelines approved by the institutional animal care and use committee of Chung-Ang University, Seoul, Korea.

Telemetric ECG

An HD-X11 heart telemetry probe (Data Sciences International, Inc., New Brighton, MN, USA) was subcutaneously implanted into the abdomen (transmitter), chest (electrode), and carotid artery (catheter) of all animals 7 days before recording an ECG to assess adaptation and recovery. After the recovery period, ECG recordings were performed on each mouse for 3 days for cardiac rhythm analysis 3 weeks after the tamoxifen injections. Eight receiver channels were used for monitoring simultaneously.

ECG

Mice were anesthetized with isoflurane (3% for induction and 1.5% for maintenance) via inhalation using a laboratory animal anesthesia machine (Vet Equipment Inc., Pleasanton, CA, USA). Following anesthesia, electrodes were inserted subcutaneously in the left and right forelimbs and left hindlimb in all animals 7 weeks after the administration of the tamoxifen injections. ECG recordings were performed using a PowerLab 4/20T with an animal BioAmp (ADInstruments, Sydney, Australia).

RNA isolation and semiquantitative or quantitative reverse transcription polymerase chain reaction analysis

Total RNA was obtained from the right atrium (RA) and SAN tissues or cells using the RNeasy® Fibrous Tissue Mini Kit (Qiagen GmbH, Hilden, Germany) and the RiboEx™ Total RNA Solution (Geneall Biotechnology Co. Ltd., Seoul, Korea). First-strand deoxyribonucleic acid was synthesized from 20 ng total RNA with random primers using a SuperScript™ III First-Strand Synthesis System (#18080051; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Polymerase chain reaction (PCR) analysis was performed using PCR Master Mix (#K0171; Thermo Fisher Scientific, Inc.) for semiquantitative reverse transcription PCR (RT‒PCR) and SsoFast™ EvaGreen® Supermix (#1725201AP; Bio-Rad Laboratories, Inc., Hercules, CA, USA) for quantitative RT‒PCR (qRT‒PCR). 18S rRNA was used as an internal control for normalization. Primers are listed in Supplementary Table 3. Postamplification melting curve analysis was performed to assess the specificity of the amplified PCR products, and relative quantities were calculated using the comparative cycle threshold method.

Western blot analysis

Western blot analysis was performed as described previously15. RA and SAN tissues or cells were lysed with lysis buffer (20 mM Tris-HCl, pH 7.4; 1% Triton X-100; 1 mM EDTA; 30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); 50 mM Na4P2O7; 100 mM NaF) with Protease Inhibitor Cocktail (Roche Life Science, Basel, Switzerland) and phosphatase inhibitors. Lysates were incubated on ice for 15 min and then centrifuged at 13,000 rpm at 4 °C for 10 min. The supernatants were used for protein quantification by the bicinchoninic acid assay, and protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 10% gel. Protein samples were transferred to a nitrocellulose membrane with a 0.45 µm pore size and blocked with 5% skim milk. Primary antibodies were diluted in blocking solution (5% bovine serum albumin (BSA) in tris-buffered saline with 0.1% Tween® 20 [anti-HCN4; #sc-58622; Santa Cruz Biotechnology, Inc., Dallas, TX, USA] dilution 1:500); anti-HCN1 (#APC-056; Alomone Labs, Jerusalem, Israel [dilution 1:1000]); anti-phospho-Akt (Ser473) ([#4060; Cell Signaling Technology Inc., Danvers, MA, USA [dilution 1:1000]); anti-Akt (#9272; Cell Signaling Technology Inc. [dilution 1:1000]), and anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) (#2118; Cell Signaling Technology Inc. [dilution 1:2000]) primary antibodies were used according to the manufacturer’s protocols. The nitrocellulose membranes were then incubated with secondary antibodies in 5% skim milk for 1 h at room temperature, and protein bands were visualized using EzWestLumi ECL solution. Densitometric quantification was performed using Bio-Rad Image Lab Software (Bio-Rad Laboratories, Inc). GAPDH was used as a loading control.

Histological analysis

Mice were sacrificed by cervical dislocation. Tissue preparation and staining were performed as described previously15,17. Isolated SAN tissues were fixed in 4% paraformaldehyde and sectioned at a thickness of 7 μm using a cryostat (Leica Biosystems, Wetzlar, Germany). Frozen SAN slides were used for hematoxylin and eosin staining, Sirius red staining, TUNEL, and immunostaining. Sirius red staining was performed by incubating the tissues in 0.1% Fast Green solution for 15 min at room temperature and then in 0.1% Fast Green solution and 0.1% Sirius red in saturated picric acid for 45 min at room temperature.

For immunohistochemical analysis of HCN4 and LC3II, peroxidase activity was blocked with 3% H2O2 in MeOH for 15 min at room temperature, blocked with 5% BSA for 1 h and treated with primary antibodies. Primary antibodies were anti-HCN4 (dilution 1:100) and anti-LC3II (#L7543; Merck KGaA, Darmstadt, Germany [dilution 1:200]). In addition, slides were incubated with biotinylated secondary antibodies and avidin–biotin complex reagents (#PK6100; Vector Laboratories, Inc., Newark, CA, USA) for 45 min at room temperature. Chromogenic detection was performed using a DAB Substrate Kit. For immunofluorescence staining of HCN4, IGF-1R, and IR, sections were blocked with 5% BSA for 1 h at room temperature, followed by overnight incubation with primary antibodies (anti-HCN4, dilution 1:50; anti-IGF-1 receptor [#sc-713; Santa Cruz Biotechnology, Inc.], dilution 1:50; and anti-insulin receptor [#AHR0271; Thermo Fisher Scientific Inc.], dilution 1:100) at 4 °C. After being washed in phosphate-buffered saline (PBS) with 0.05% Triton X-100 (PBST) for 10 min each, the slides were incubated with secondary antibodies (Alexa Fluor™ 647-conjugated goat anti-rat IgG, Alexa Fluor™ 488-conjugated goat anti-rabbit IgG, Alexa Fluor™ 488-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Inc.) for 1 h at room temperature. After being washed with PBS, the slides were mounted with diamidino-2-phenylindole (DAPI) solution. Images were obtained using a confocal microscope (LSM 700; Carl Zeiss AG, Jena, Germany). Apoptosis was determined using a colorimetric DeadEnd™ TUNEL Assay Kit (#G7130; Promega Corp., Madison, WI, USA) following the manufacturer’s instructions.

Isolation of SAN pacemaker cells and immunocytochemistry

Mice were anesthetized with isoflurane to extract SAN tissues, and then pacemaker cells were isolated as previously described18. The SAN tissues were chopped into 4–5 pieces and incubated in a low-Ca2+ solution at 36 °C for 5 min. The low-Ca2+ solution contained 140 mM NaCl, 5 mM HEPES, 5.5 mM glucose, 5.4 mM KCl, 0.2 mM CaCl2, 0.5 mM MgCl2, 1.2 mM KH2PO4, 50 mM taurine, and 1% BSA; the pH was adjusted to 6.9 with NaOH. The SAN tissues were then incubated in 3 ml of enzyme solution containing collagenase type II (Worthington Biochemical Corp., Lakewood, NJ, USA), elastase (Worthington Biochemical Corp.), and protease type XIV (Sigma‒Aldrich®, Merck KGaA, Darmstadt, Germany) in a low-Ca2+ solution for 15–20 min. The tissue was then exposed to Kraft-Brule solution at room temperature and slowly dissociated using a wide-diameter glass pipette for 3–5 min. The dissociated cells were plated on laminin-coated coverslips (Supplementary Fig. 4). Subsequently, the samples were immunocytochemically stained or stored at 4 °C to record the If. For immunocytochemical analysis, attached SAN pacemaker cells were fixed with 4% paraformaldehyde at room temperature for 10 min, permeabilized with 0.1% PBST for 10 min, blocked with 5% BSA, and incubated overnight at 4 °C with anti-HCN4 antibody (dilution 1:50). After being washed three times in PBS for 10 min each, the coverslips were treated with secondary antibodies (Alexa Fluor™ 488-conjugated goat anti-rat IgG) for 1 h at room temperature and then mounted with DAPI solution. Images were obtained using a confocal microscope.

Patch-clamp electrophysiology

The If was recorded as previously described19,20. Whole-cell voltage-clamp recordings were carried out in isolated pacemaker cells from the SAN. The extracellular solution contained the following: 140 mM NaCl, 5.4 mM KCl, 1.2 mM KH2PO4, 5 mM HEPES, 5.5 mM glucose, 1 mM MgCl2, and 1.8 mM CaCl2. The pH was adjusted to 7.4 with NaOH. The current was elicited with a holding potential at −50 mV and a hyperpolarizing step to −110 mV for 1.0 s. The patch pipettes were filled with intracellular solution containing 5 mM NaCl, 135 mM KCl, 10 mM HEPES, 1 mM MgCl2, 0.1 mM CaCl2, 10 mM EGTA, 4 mM MgATP, and 5 mM pCr; the pH was adjusted to 7.2 with KOH. After the initial current recording, 0.5 mM BaCl2 was added to the extracellular solution to eliminate the Ba2+-sensitive K+ current. The current density was calculated by dividing pA by pF.

Isolation of neonatal rat cardiomyocytes

Neonatal hearts were obtained from three-day-old Sprague Dawley rats and digested for isolation of cardiomyocytes as previously described21. Neonatal rat cardiomyocytes (NRCMs) were incubated in DMEM/M199 (1:1) containing 10% horse serum, 5% fetal bovine serum, 1% L-glutamine, 1% bromodeoxyuridine, and 1% penicillin/streptomycin. To confirm the relationship between insulin and HCN channel expression, NRCMs were treated with 200 nM insulin (#I9278; Merck KGaA, Darmstadt, Germany) with or without the PI3K inhibitor LY294002 (#L9908; Merck KGaA, Darmstadt, Germany) for 6–24 h and then analyzed by western blotting and qRT‒PCR.

Echocardiography

Mice were anesthetized in an induction chamber using 3% isoflurane, and anesthesia was maintained with 1.5% isoflurane on a heating pad at 36 °C. Echocardiography was performed using the Vevo 770 System (FUJIFILM VisualSonics Inc., Toronto, ON, Canada) with a 30-MHz transducer in the 2-dimensional and motion-mode images22.

Statistical analysis

Data are presented as the mean ± SEM. For intergroup comparisons, the data distribution was first evaluated for normality using the Shapiro‒Wilk test, a further Shapiro‒Wilk test after log-transformation, and a quantile‒quantile plot. The normally distributed data were compared using a t test or two-way analysis of variance. The nonnormally distributed data were analyzed using either an unadjusted Mann‒Whitney U-test or a Mann‒Whitney U-test with a Bonferroni-corrected α value. P < 0.05 was considered statistically significant. All analyses were conducted using the Statistical Package for the Social Sciences (SPSS®, Version 23, IBM Corp., Armonk, NY, USA).



Source link

Related Articles

Leave a Reply

Stay Connected

9FansLike
4FollowersFollow
0SubscribersSubscribe
- Advertisement -spot_img

Latest Articles

%d bloggers like this: