Guinea-pig ventricular myocytes

Primary guinea pigs’ ventricular cardiac myocytes (used in electrophysiological and biochemical assays) were isolated as previously described [20, 32–34]. Briefly, adult male and female Hartley guinea pigs were deeply anesthetized with isoflurane in accordance with the IACUC approval of this study at the VA New York Harbor Healthcare System and conforming to the NIH guidelines. Hearts were excised, Langendorff perfused with Tyrode solution containing (in mM): 118 NaCl, 4.8 KCl, 1 CaCl₂, 10 Glucose, 1.25 MgSO₄, 1.25 K₂HPO₄ (pH = 7.4) for 5 minutes. The heart was then perfused with Ca²⁺-free Tyrode solution for 10 minutes before switching to Ca²⁺-free Tyrode solution containing Collagenase B (final concentration, 0.6 mg/ml; Boehringer Mannheim, Indianapolis, IN) for an additional 6 minutes. The heart was subsequently perfused with high-K solution containing (in mM): 70 KOH, 50 L-glutamic acid (potassium salt), 40 KCl, 10 Taurine, 2 MgCl₂, 10 Glucose, 10 HEPES, 5 EGTA, and 1% albumin (pH 7.4, with KOH) for 5–10 minutes. The digested heart tissue was placed in fresh high-K solution, minced into smaller pieces and triturated several times to dissociate the cells. The cell suspension was filtered through a mesh and allowed to settle for 15–20 min. The pellet was resuspended in 10% M199 media and plated on laminin-coated coverslips. Cells were patched 6–8 hours after plating. The external solution used for I_Kr and I_K1 recordings contained (in mM): 145 NaCl, 4.5 KCl, 1 MgCl₂, 1.8 CaCl₂, 10 HEPES, and 10 glucose (pH 7.4). Ca currents were blocked by the addition of 5 μM nifedipine in the bath solution and the slow delayed rectifier K current (I_Ks) was blocked with 100 μM chromanol. The pipette solutions for recording I_Kr and I_K1 contained (in mM): 140 KCl, 10 HEPES, 11 EGTA, 1 MgCl₂, 1 CaCl₂, 5 MgATP, and 5 K₂ATP; the pH adjusted to 7.2 with KOH. Currents were recorded in the whole-cell, voltage clamp configuration of the patch-clamp technique using an Axopatch-200B amplifier (Axon Instruments, Inc., Burlingame, CA). I_Kr was recorded using a short 200 ms depolarizing pulse from a holding potential (HP) of -50 mV and test pulses were applied at various voltages from -40 to +80 mV in a 10 mV increment before returning to -40 mV for tail current recording. I_K1 was activated from -80 mV to test potential ranging from -120 mV to +10 mV in 10 mV steps for 200 ms. The external solution for I_Na recordings contained (in mM): 20 NaCl, 5 CsCl, 115 tetraethylammonium chloride (TEACl), 1 MgCl₂, 10 HEPES, and 10 glucose (pH 7.4, with CsOH). L-type Ca current and T-type Ca current were blocked by CoCl₂ (5 mM) and NiCl₂ (1 mM), respectively. The internal solution contained (in mM): 140 CsCl, 10 NaCl, 3 MgCl₂, 5 EGTA, 10 HEPES, and 2 MgATP (pH 7.2, CsOH). I_Na was evoked from -80 mV to test potentials ranging from -70 mV to +20 mV in 10 ms steps for 30 ms. Action potentials were recorded from single ventricular myocytes in current-clamp mode by passing depolarizing currents at subthreshold (1.4 X) intensity. Data were sampled with an A/D converter (Digital 1320A, Axon Instruments) and stored on the hard disk of a computer for subsequent analysis. Currents were sampled at 20 kHz and filtered at 5 or 10 kHz. Traces were acquired at a repetition interval of 10 s.