Embryonic cardiomyocyte isolation and culture
Pregnant female mice were killed by cervical dislocation and embryos, at different stages of development (11-13 days post coitus: early stage; 14-16 days: intermediate stage; or 17-20 days: late stage), were removed. For neonatal stage myocytes, mice at 1 day post partum were killed by decapitation, just prior to the removal of cardiac tissue as described by Sturm & Tam (1993) and in accordance with the guidelines of the local ethics committee (Institutional Animal Care and Use Committee, Columbia University College of Physicians and Surgeons). Cardiac myocytes were isolated from embryonic and neonatal hearts as described previously (Kubalak et al. 1995). In brief, hearts were dissected from embryos and neonates and placed in normal Tyrode solution (Kass et al. 1989). Atrial and ventricular tissues were separated under a dissecting microscope and placed in an Eppendorf tube with 0.5 ml Tyrode solution containing 0.5 mg ml−1 collagenase Type II (Worthington) and 1.0 mg ml−1 pancreatin (Gibco) for a 15 min digestion at 37°C. Cells from the enzymatic digestion were placed in culture medium (modified Eagle's medium; Gibco) containing 10 % fetal bovine serum, plated into plastic Petri dishes and cultured in a 10 % CO2 incubator at 37°C. Electrophysiological recordings were carried out approximately 18-24 h after plating of the cells and could be carried out for periods up to 48 h following plating. Unless specified for individual experiments, cells were maintained in agonist-free media for this entire period.
Experimental results shown in this paper were obtained using patch clamp procedures in conventional whole-cell (Hamill et al. 1981) or in the perforated patch configuration (Horn & Marty, 1988). In experiments to study IK,s, E4031 (5 μM; obtained as a gift from Eisai Limited, Tokyo, Japan) was added to the external solution to block the rapid component of the delayed rectifier K+ channel current (Sanguinetti & Jurkiewicz, 1990) and nisoldipine (1 μM) was added to block the L-type Ca2+ channel current. For the measurement of IK,s, the external solution contained (mM): KCl, 5; N-methyl-glucamine, 125; MgCl2, 1; CaCl2, 1; Hepes, 10; glucose, 5 (pH 7.4 with KCl). The internal solution for recording whole-cell IK,s contained (mM): potassium aspartate, 110; CaCl2, 1; Hepes, 10; EGTA, 11; MgCl2, 1; K2ATP, 5; pH 7.3 (KOH). Whole-cell ICa was recorded using external solution containing (mM): CsCl, 5; Hepes, 10; N-methyl-glucamine, 125; glucose, 5; MgCl2, 1. BaCl2 (20 mM) was added to this solution as charge carrier and the internal solution contained (mM): aspartic acid, 50; K2ATP, 5; CsCl, 60; EGTA, 11; Hepes, 10; CaCl2, 1 (pH 7.2 with CsOH). For perforated patch recordings of L-type Ca2+ channel currents, amphotericin B was dissolved in DMSO at a concentration of 30 mg ml−1, and then added to the above internal solution to yield a final concentration of 120-250 μg ml−1. Both the amphotericin B stock solution and the amphotericin B-containing pipette solution were subjected to 5-10 min of ultrasonication before use. Capacity transients were monitored as a function of time after attaining a high resistance seal with the surface membrane. Electrical access to the cell was judged by the time course of the capacity transient, and adequate access was usually attained within 10 min of seal formation.
In order to measure the time course of regulatory responses, Ca2+ channel currents were measured during test pulses (40 ms) to +10 mV applied once every 10 s. Holding potentials of -40 mV were used for both Ca2+ and K+ channel current recordings, and isochronal (2 s pulses applied at 10 s intervals) activation curves were used to measure the activation of IK,s. Similarly, ICa activation was measured by 40 ms test pulses to a series of potentials (10 mV increments) applied at 10 s intervals from a holding potential of -40 mV to +60 mV. ICa inactivation curves were obtained by measuring peak current at a test potential of +10 mV after application of a series of 5 s conditioning pules (-80 to +30 mV, 10 mV increments). A 10 ms return to the holding potential (-40 mV) separated each test and conditioning pulse. Patch pipettes (Clay Adams glass) were pulled to resistances of 2.5-5.0 MΩ when filled with internal solution. Total cell membrane capacitance was used as a measure of membrane area and was determined either by analog capacity compensation or by integration of current transients in response to 10 mV test pulses. Electrophysiological recordings were carried out at room temperature (20-22°C) except for the experiments in which the slow delayed rectifier K+ channel was transiently exposed to 8-Br-cAMP, which were conducted at 30-32°C.
Chemicals were obtained from the following suppliers: amphotericin B (Sigma); 8-chlorophenylthio-cAMP (8-CPT-cAMP; Boehringer Mannheim); 8-Br-cAMP (Sigma); adenosine 3′,5′-cyclic monophosphothioate, RP-isomer, triethylammonium salt (Rp-cAMPS; CalBiochem). Stock (20 mM) solutions of 8-CPT-cAMP or 8-Br-cAMP (dissolved in H2O) were mixed and diluted to 300 μM for each experiment. Stock Rp-cAMPS solution (10 mM dissolved in H2O) was mixed and diluted daily to 300 μM.
Data were collected, stored and analysed on IBM (486)-compatible computers interfaced to Axopatch (200A) amplifiers (Axon Instruments) under the control of pCLAMP (version 6.0) software (Axon Instruments). Graphics and statistical data analysis were carried out using Origin software (Microcal, Northampton, MA, USA). Averaged data are shown as means ±s.e.m. and were compared using Student's t test with a P value of < 0.05 taken to indicate statistical significance.
Cell capacitance was measured and compared for TG+ and transgenic negative (TG-) cells as a function of developmental stage. For each stage there was no significant difference at the 0.05 level between TG+ and TG- cell capacitance; data obtained were (TG-, TG+, means ±s.e.m.): early stage: 25.4 ± 3.3 pF (n= 10), 29.9 ± 6.5 pF (n= 8); intermediate stage: 23.4 ± 7.5 pF (n= 26), 26.0 ± 9 pF (n= 34); late stage: 26.5 ± 11.5 pF (n= 32), 31.4 ± 3.2 pF (n= 15); neonatal: 25.7 ± 1.8 pF (n= 10), 28.0 ± 3.2 pF (n= 20).