The intact hearts were dissected out quickly from the anaesthetized mice and were perfused with freshly made perfusion buffer with liberase TH as described before (Givvimani et al. 2015 (link)). The yield was 80–85% and did not vary between the groups. The isolated myocytes were used immediately for the contractility measurements, calcium transits, flow cytometry analysis, mitochondrial fractionation and oxygen consumption rates (OCR) measurements. The systolic and diastolic on isolated myocytes were recorded by Ion-Optics (Boston). The decay of calcium transient was also recorded as previously described (Veeranki et al. 2016 (link)). A sub set of cardiomyocytes were incubated with Fura-2-AM (1.0 μmol/l) for 30 min, and fluorescence measurements are recorded with a dual-excitation fluorescence photomultiplier system (IonOptix) as described before after 1 Hz field stimulation (Givvimani et al. 2015 (link)). Myocytes were field stimulated (at a frequency of 1.0 Hz, pulse duration of 4 ms and amplitude of 10 volts) using IonOptix myopacer and the contractions were recorded through SoftEdge™ Acquisition Software as described before (Givvimani et al. 2015 (link)). A batch of 5 randomly-selected myocytes were recorded for the contraction parameters at a time from an unstimulated pool and a total of 20 myocyte recordings per heart were collected for further analysis.
Myopacer
Manufactured by IonOptix
Sourced in United States
The MyoPacer is a laboratory instrument designed to stimulate muscle tissue samples. It provides electrical stimulation to elicit and measure the contractile responses of muscle samples under controlled conditions.
Automatically generated - may contain errors
Lab products found in correlation
Fura 2 am, by Thermo Fisher Scientific (8 mentions)
Ionwizard software, by IonOptix (6 mentions)
Ionwizard, by IonOptix (6 mentions)
Contractility system, by IonOptix (4 mentions)
Myocam, by IonOptix (4 mentions)
Eclipse ts100, by Nikon (3 mentions)
Ix73 inverted microscope, by Hamamatsu Photonics (2 mentions)
Flash 4orca, by Cairn Research (2 mentions)
Field stimulator, by IonOptix (2 mentions)
Detach 2 kit, by PromoCell (2 mentions)
Dl isoproterenol hydrochloride, by Merck Group (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Ionwizard 6, by IonOptix (2 mentions)
Dual excitation fluorescence photomultiplier system, by IonOptix (1 mentions)
Prl tk renilla luciferase construct, by Promega (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Sp8 sted confocal microscope, by Leica (1 mentions)
Isoproterenol, by Merck Group (1 mentions)
Smz745t, by Nikon (1 mentions)
Cl 100 temperature controller, by Warner Instruments (1 mentions)
48 protocols using myopacer
1
Isolation and Analysis of Murine Cardiomyocytes
2
Investigating Transcriptional Regulation in NRVMs
3
Cardiomyocyte Pacing Protocol
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To provide a constant stimulus frequency the cardiomyocytes were field stimulated at 1Hz using customised graphite electrodes (15V, 2ms pulse ~ 20% above threshold) at a fixed rate using a MyoPacer (IonOptix Corp, Dublin, Ireland). Pacing energy was adjusting by varying the voltage amplitude of the stimulus while keeping the duration and current constant.
4
Contractility Measurement in Myocytes
5
Calcium Sparks in Isolated Myocytes
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The calcium spark was detected in isolated myocytes by confocal optical calcium imaging according to the methods described previsouly.17 Isolated myocytes were loaded with 10 μmol/L Fluo3/AM for 30 min at 37°C in dark. After washing by PBS for two times, calcium sparks were observed with an SP8 STED confocal microscope (Leica) equipped with an argon laser at wave length at 488 nm. Line scan was used to acquire the line scan images (512 pixels per line) at sampling rate of 2 ms per line. The scanning frequency was 600 Hz. Calcium sparks were analysed by ImageJ with SparkMaster Plugin according to previously described method.18 Calcium transients were triggered by MyoPacer (IonOptix) at 0.5 Hz and 40 V at 23°C. Calcium re‐uptake was assessed by calculating calcium removal time constant (Tau) by using IonWizard software (IonOptix). Cell shortening was measured by using an edge detection system (Crescent Electronics). Cell shortening was presented as percentage of contractile cell length/resting cell length.
6
Cardiomyocyte Contractility and Calcium Dynamics
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Cardiomyocyte contractility and Ca2+ transients were recorded as described (Guan et al., 2015 (link)). Briefly, the cell suspension was gently added dropwise to the perfusion chamber of the inverted microscope. After a few minutes of sedimentation, the cells were perfused with Ca2+-free Tyrode’s solution with 1.8 mM CaCl2 at a rate of 1 ml/min. The contraction of myocardial cells was measured by the IonOptix Myocam assay system (IonOptix, Milton, MA, USA) and field stimulation was given at 0.5 Hz (duration 2-ms) using a cell stimulator (MyoPacer, IonOptix).
Ca2+ transients were detected simultaneously with contraction of the cardiomyocytes. Experimental steps were performed in the dark due to the light sensitivity of the fluorescent Ca2+ indicator. Cardiomyocytes were loaded with fluorescent Ca2+ indicator fluo2-AM (2 mM) for 10 min. Cells were stimulated with a field intensity of 0.5 Hz and passed alternately through 340 and 380 nm filters (bandwidth of ±15 nm). Finally, the emitted fluorescence was observed at 510 nm.
Ca2+ transients were detected simultaneously with contraction of the cardiomyocytes. Experimental steps were performed in the dark due to the light sensitivity of the fluorescent Ca2+ indicator. Cardiomyocytes were loaded with fluorescent Ca2+ indicator fluo2-AM (2 mM) for 10 min. Cells were stimulated with a field intensity of 0.5 Hz and passed alternately through 340 and 380 nm filters (bandwidth of ±15 nm). Finally, the emitted fluorescence was observed at 510 nm.
7
Traction Force Microscopy of Engineered Cardiac Tissues
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Traction force microscopy experiments were conducted as previously described [7 (link), 8 (link), 10 (link)]. On day 4 of culture, cardiac mTissues in modified Tyrode’s solution (1.8 mM CaCl2, 5 mM glucose, 5 mM HEPES, 1 mM MgCl2, 5.4 mM KCl, 135 mM NaCl, 0.33 mM NaH2PO4, pH 7.4) were imaged on an environmental controlled line scanning confocal microscope (Zeiss LSM510) using a 40X air objective (with a 0.5x zoom) or a 20x objective to ensure the full diamond-shaped tissue fit within the diagonal of the field of view. Movies of contracting myocytes and bead displacement were imaged at 33 Hz with both brightfield, and 488 laser excitation and recordings were performed over multiple [6 –10 (link)] contractile cycles. Differently, than single-cell or cell-pair assays, mTissues could be electrically paced to control the beating rate using two custom-made platinum electrodes inserted onto the lid of a 35mm petri dish and connected to an external field stimulator (Myopacer, IonOptix Corp., Milton, MA). Recordings were conducted at 2 Hz and using voltages between 8 to 11 V that reliably overdrove spontaneous beating in the mTissues.
8
Langendorff-Perfused Mouse Heart Arrhythmia Induction
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At 1 and 8 weeks after MI, mouse hearts were isolated and Langendorff-perfused with Tyrode solution (37 °C) containing 1 µM isoproterenol (Sigma, Catalogue No.: I6504), ex vivo ECG were continuously measured by placing recording electrodes around the heart as we previously described23 (link),54 (link) (Fig. 1 E). PES was applied using a MyoPacer (IonOptix) via a pair of platinum electrodes placed on the left ventricular apex of the heart. The standard stimulation protocol (Fig. 1 E) consisted of 10 stimuli at 100 ms intervals (S1, 5V) followed by one extra stimulus (S2) starting at an interval of 80 ms which was then reduced by 2 ms until the effective refractory period (ERP) was reached. If VT or VF was not induced, a second extra stimulus (S3) was added at 80 ms after S2. The S3 interval was then reduced by 2 ms until the ERP was reached. Finally, a third extra stimulus (S4) was added 80 ms after S3 and was then decreased by 2 ms until the ERP was reached. If a heart failed to develop a VT or VF with 3 extra stimuli, the heart was deemed non-inducible.
9
GCamP6f Calcium Imaging in Cardiomyocytes
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GCamP6f was kindly provided by Professor Bruce Conklin (Gladstone Institutes) and differentiated as outlined in section 2.6 and recorded in Tyrode’s solution. To create this solution, the following were added to 1 l of dH2O while mixing using a magnetic stirrer: 8.18 g of NaCl (140 mM), 0.3375 g of KCl (6 mM), 1.86 g of glucose (10 mM), 2.38 g of HEPES (10 mM), 1 ml of 1 M MgCl2 solution (1 mM) and 1.8 ml of 1 M CaCl2 solution (1.8 mM) and adjusted with 2 M NaOH to pH 7.4. The samples were kept under superfusion of 37 °C Tyrode’s solution. The cells were stimulated at 1 Hz using MyoPacer (IonOptix) The records were obtained using a Nikon IX73 Inverted Microscope equipped with a Hamamatsu Flash 4 Orca and LED illumination (Cairn Research) by WinFluor software (University of Strathclyde). Raw data from WinFluor were preprocessed using Fiji image analysis software to obtain a fluorescent intensity profile. The data were analyzed by a blinded investigator. The records were analyzed using a custom MATLAB® code (N = 4, n ⩾ 7).
10
Differentiation and Imaging of GCamP6f
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