Aerobic interval training reduces inducible ventricular arrhythmias in diabetic mice after myocardial infarction
NataleRolim Kristine Skårdal Morten HøydalMirta M. L. Sousa Vegard Malmo GuriKaurstad Charlotte B. IngulHarald E. M. Hansen Marcia N. AlvesMarteThuen Olav Haraldseth Patricia C. BrumGeirSlupphaug Jan PålLoennechen Tomas StølenUlrikWisløff
Online Appendix
Surgical procedure
Prior to surgery, buprenorphine was subcutaneously administered (0.1 ml 0.03 mg/ml Temgesic, Reckitt and Coleman, Hull, UK) to reduce post-operative acute pain. Following a 30-minute period, the mice were anaesthetized with 5 % isoflurane in O2, intubated and connected to a rodent ventilator (Harvard Starling ‘Ideal’ Ventilator, Sydney, Australia), with a stroke volume of 0.4 ml/stroke and a respiration rate of 80-85 strokes/min. A steady level of anesthesia was ensured by delivery of 2 % isoflurane. The chest was then opened and the heart visualized, the pericardium was ruptured and infarction was induced by permanent ligation of the left anterior descending artery. Occlusion was confirmed by the rapid change in color and restricted ventricular motion. The sham group had the pericardium ruptured before the chest was closed. A single measurement from the middle level of the left ventricle (LV) was used to estimate the size of myocardial infarction (MI) by percentage of LV perimeter occupied by the akinetic region [2]. Only animals that survived 24 hours after surgery and with MI size between 35 and 45 % of the LV were included in this study. The Online Appendix presents the technical details on animal surgery.
Magnetic resonance imaging (MRI)
LV volumes were assessed using MRI (7T Bruker BiospecAvance 70/20, Bruker Biospin, Ettlingen, Germany). A 72 mm volume coil were used for transmission and an actively decoupled rat head surface coil for receive only (RAPID MR International, LLC, Ohio, USA). Anesthetized animals were positioned prone atop of the surface coil in a dedicated animal bed. A steady state of anesthesia was maintained using 1-2 % isoflurane in 1:2 O2:N2 delivered through a face mask and kept at 80-120 breaths/min. Rectal temperature was kept stable at 36-38°C by a negative feedback system with heated air. Contiguous 1 mm short axis black blood cine movies were acquired using a self-gated fast low angle shot sequence using the Bruker IntraGate macro. Other acquisition parameters were repetition time = 6.86 ms, echo time = 2.18 ms, flip angle = 10°, field of view = 4 × 4 cm2, 300 repetitions and matrix = 128 × 128, resulting in an in-slice pixel size of 0.3125 × 0.3125 mm2. 6-9 slices were acquired for coverage of the complete LV. Following acquisition, end-diastolic and end-systolic volumes were extracted using a specifically designed texture based semi-automatic segmentation procedure.
Echocardiography
Cardiac function was assessed using echocardiography including pulsed Doppler and tissue Doppler imaging (Vevo 770 high-resolution in vivo imaging system, VisualSonics, Toronto, Canada). An apical four-chamber view was used to identify the mitral valve and the Doppler flow spectra recorded at the center of the left atrioventricular orifice. Early diastolictransmitral peak flow velocity (E velocity) was identified. Tissue Doppler imaging was performed at the septal corner of the mitral annulus and the peak systolic tissue Doppler velocity (Sa) and peak early tissue Doppler velocity (Ea).
Adult mice cardiomyocytes isolation
At the end of the in vivo experiments, mice hearts were heparinized and rapidly excised under a mixture of 3-4 % isoflurane and 96-97 % oxygen. Until it was connected to the aortic cannula on a standard Langendorff retrograde perfusion system, the hearts were kept on ice coldcalcium free Krebs-solution. Cardiomyocytes were dissociated using Krebs-Henseleitcalcium free solution and collagenase type 2 (Wortington, Lakewood, USA), as previously described [4]. After the hearts were taken down, left ventricles were carefully cut off and gently shaken for 2 min before the non-digested tissue was filtered out (nylon mesh, 250 µm). Further, cells were gently introduced tocalcium and kept at 0.6 mMcalcium until experiments were conducted.
Cardiomyocytes shortening and Ca2+ handling
Fura-2/AM-loaded (2 µmol/L, Molecular Probes, Eugene, OR, USA) cardiomyocytes were stimulated by bipolar electrical pulses at 2 Hz on an inverted epifluorescence microscope (Nikon TE-2000E, Tokyo, Japan), whereupon cell shortening was recorded by video-based myocyte sarcomere spacing (SarcLen™, IonOptix Corporation, MA, USA). Intracellular calcium concentration was measured by exciting Fura-2 at 340 and 380 nm and collecting the emission ratio at 510 nm with a PMT Acquisition Add-on module (PMTACQ, IonOptix). During the stimulation protocol, cells were continuously perfused with normal physiological HEPES - based solution (135 mM NaCl, 5 mMKCl, 1 mM MgCl2.6H2O, 1.8 mM CaCl2.2H2O, 10 mM HEPES, 8 g/liter glucose-H2O, and pH adjusted to 7.4 with NaOH).
Quantitative measurements of calcium removal during diastole
The sarcoplasmic reticulum (SR) calcium ATPase (SERCA2a) and Na+/Ca2+ exchanger (NCX) may contribute to removal of calcium from cytosol during diastole [1]. We used the rate constant of calcium decline in three different solutions to quantify the contribution from each factor: a) rate constant of calcium decline during electrical stimulation in normal HEPES 1.8 mMcalcium solution with all contributors active, b) addition of 10 mM caffeine in normal HEPES 1.8 mMcalcium solution that inhibits the effect of SERCA2a and c) addition of 10 mM caffeine in a 0Na+/0Ca2+ solution that inhibits both SERCA2a and NCX.
Quantitative measurements of diastolic calcium leak
On an inverted epifluorescence microscope, the method of Shannon et al. [3] was used to determine SR diastolic calcium leak. To bring the cellular calcium content to a steady state, we stimulated the cardiomyocytes electrically at 1 Hz in normal HEPES - based 1.8 mM [Ca2+]-solution for 30-60 seconds. After the last electric stimulus, we rapidly switched the perfusion to a 0Na+/0Ca2+ containingsolution and measured diastolic intracellular calcium concentration([Ca2+]i) in quiescent non-stimulated cardiomyocytes. The resting period lasted for one minute. The 0Na+/0Ca2+ solution prevents the NCX, which is the primary calcium influx and efflux mechanism at rest. Tetracaine blocks the calcium leak over the RyR. The quantitative difference between diastolic [Ca2+]i with and without tetracaine unveils leakage. We performed the measurements in presence and without tetracaine (1 mM). After the one-minute period in 0Na+/0Ca2+ solution, we added caffeine (10 mM) to assess the SR calcium content. Diastolic SR calcium leak is presented as diastolic calciumin relation to total SR calcium content. To control for protein kinase A (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII) effects on SR calcium leak, we incubated cells with the PKA inhibitor (H-89 3 µM, Sigma Aldrich) and CaMKII inhibitor (autocamtide-2 related inhibitory peptide, AIP 1 µM, Sigma Aldrich) in a subset of experiments.
Western-blotting analysis
The expression of calciumhandling proteins was analysed in LV homogenates. Proteins were heated in LDS loading buffer (Invitrogen) prior to separation on pre-cast 4-12 % denaturing NuPAGE gels using MOPS running buffer or 3-8 % Tris-acetate denaturing NuPAGE gels using Tris-acetate running buffer (Invitrogen). Proteins were then transferred to PVDF membranes (Immobilon-FL, Millipore) for 2 h at 30 V after separation on 4-12 % gels or transferred for 20 h at 20 V at 4°C after separation on 3-8 % gels. Western blotting analysis were performed using polyclonal anti-SERCA2a (1:5,000; A010-20, Badrilla, Leeds, UK), monoclonal anti-NCX (1:1,000; MA3-926, Thermo Fisher Scientific, Rockford, IL, USA), monoclonal anti-RyR (1:5,000; MA3-925, Thermo Fisher Scientific), monoclonal anti-CAMKII delta (1:200, ab54927, Abcam, Cambridge, UK), polyclonal anti- Cav1.2 alpha 1c (voltage-gated calciumchannel subunit alpha Cav1.2) (1:200; ACC-003, Alomone labs, Jerusalem, Israel). Monoclonal anti- glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:10,000; G9295, Sigma, St Louis, MO, USA), and polyclonal anti--tubulin (1:1,000; ab21058, Abcam) were used to normalize the data. After incubation with goat anti-mouse IRDye800LT and goat anti-rabbit IRDye680LT secondary antibodies (Li-COR), bands were detected on an Odyssey infrared imaging system (Li-COR) and quantitative analysis performed by Odyssey v.3.0 software.
References
1.Bers DM (2002) Cardiac excitation-contraction coupling. Nature 415:198-205 doi:10.1038/415198a
2.Dos Santos L, Mello AFS, Antonio EL, Tucci PJF (2008) Determination of myocardial infarction size in rats by echocardiography and tetrazolium staining: correlation, agreements, and simplifications. Braz J Med Biol Res 41:199-201 doi:10.1590/S0100-879X2008005000007
3.Shannon TR, Ginsburg KS, Bers DM (2002) Quantitative assessment of the SR Ca2+ leak-load relationship. Circ Res 91:594-600 doi:10.1161/01.RES.0000036914.12686.28
4.Wisloff U, Loennechen JP, Currie S, Smith GL, Ellingsen O (2002) Aerobic exercise reduces cardiomyocyte hypertrophy and increases contractility, Ca2+ sensitivity and SERCA-2 in rat after myocardial infarction. Cardiovasc Res 54:162-174 doi:10.1016/S0008-6363(01)00565-X
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