Gerrit Isenberg

Calcium tolerant ventricular myocytes prepared by preincubation in a “KB medium”

Abstract1.Ventricular myocytes were isolated by means of collagenase and hyaluronidase. Hearts of rats or guinea pigs were perfused in a retrograde manner; bovine ventricular tissue was incubated in from of tissue chunks.2.In “low Ca medium” (aCa=1 μM), the myocytes beat spontaneously. Resting potentials of −4 mV and input resistances of 1 MΩ, associated with a fast decay of aKi, indicated “hyperpermeability” of the cell membrane. Within 2 h, the cells aged: they granulated (swelling of mitochondria), contracted, rounded up and finally disaggregated. When the superfusate was changed to Tyrode solution (3.6 mM CaCl2), 90–98% of those cells responded with the “Ca paradox”. Initially, aCai increased from 0.2 μM to >10 μM, and the sarcomere length (SL) shortened to <1.5 μm. Within the following 3 min aCai renormalized but the contracture was sustained. After another 5 min, the SL shortened to <1.2 μm, but a delay of 1–2 min passed before aCai started to increase towards aCa0. The 2–10% Ca tolerant cells beat spontaneously. Their resting potentials were between −10 and −70 mV, their input resistances between 4 and 20 MΩ. Repolarization by anodal current flow could restore the sodium spike but not the plateau of the action potential.3.Freshly prepared myocytes were pre-incubated in a KB medium composed of 85 mM KCl, 30 mM K2HPO4, 5 mM MgSO4, 5 mM Na2ATP, 5 mM pyruvate, 5 mM succinate, 5 mM β-OH-butyrate, 5 mM creatine, adjusted with KOH to pH 7.2 and with EGTA to pCa 7.5. The minimum time of preincubation was 1 h, the maximum 5 days when the cells were stored at 5°C. The conditioning with the KB medium improved both Ca tolerance and electrophysiology significantly. About 70% of the rod shaped myocytes were Ca tolerant. These cells had resting potentials between −75 and −90 mV, the membrane resistances were around 7 kΩ×cm2. The action potentials showed well pronounced plateaus and lasted for 125 ms (rat), 300 ms (guinea pig) or 400 ms (bovine). Their shape showed the typical species related pecularities.4.We discuss that both ageing and Ca intolerance of the un-conditioned cells result from Ca overload through the hyperpermeable membrane, and that Ca overload causes ATP depletion via mitochondrial disfunction. The constituents of the KB medium may act to prevent these processes.

Calcium currents of isolated bovine ventricular myocytes are fast and of large amplitude

Abstract1.A two microelectrode voltage clamp technique was applied to ventricular myocytes superfused with Tyrode solution containing 1.8 or 3.6 mM [Ca]0. The clamp settled a 100 mV step up over a “capacitive membrane area” of 2×10−4 cm2 within 200 μs; the capacitive current peaked within 60 μs and decayed afterwards with a τc of 60 μs, indicating a non-distributed series resistance of less than 30 Ohm·cm2.2.Clamping from resting potential (−80 mV) to 0 mV evoked the 2 inward current componentsINa andICa.INa was greater than 50 nA and prevented adequate voltage control during the initial 2 ms; it could be blocked by 60 μM TTX, sodium removal or by clamping from a conditioning pre-step of −50 mV.ICa remained essentially unaltered by the 3 procedures listed above, but could be blocked by 2 μM D600, by 5 mM Ni or by 5 mM Co.3.Clamping from −50 to 0 mV evoked a net inward current which peaked within 2 ms to −8 nA and changed 200 ms later into a net outward current. Plotting the time dependent values on semilog paper, 3 time constants became apparent. From tail current analysis and sensitivity to D600 we attribute the slow exponential (τ≈1s) to activation of potassium current (Ix), and the 2 faster exponentials (τ ≈ 1 s) to inactivation ofICa.4.ICa was defined by the time course of inactivation. At 0 mV, it had a peak amplitude of 34±12 μA/cm2 in 1.8 mM [Ca]0. Doubling [Ca]0 to 3.6 mM increased peakICa to 42±10 μA/cm2. Addition of 0.2 μM adrenaline increased peakICa up to 80 μA/cm2. The estimates of peakICa were about 20% greater whenICa was defined by its sensitivity to D 600.5.Voltage dependence.ICa had a threshold at about −35 mV, reached a maximum around +5 mV and declined again for more positive potentials. Between +50 and +60 mV the peak changed from a (net) inward to a net outward current; this could indicate an “apparent” reversal potential (Erev) but also masking ofICa by a transient outward current.6.Activation. By dividing peakICa by (V-Erev) the steady-state activation curve was estimated. The data could be fitted according to

Action potentials and net membrane currents of isolated smooth muscle cells (urinary bladder of the guinea-pig)

\bar g/(1 + exp(V - V_h )/k)

The shortening of the action potential by DNP in guinea-pig ventricular myocytes is mediated by an increase of a time-independent K conductance

using

Calcium currents of cesium loaded isolated smooth muscle cells (urinary bladder of the guinea pig)

\bar g = 0.76 \pm 0.25 mS/cm^2

Cardiac Purkinje fibres

,k=9±1.5 mV andVh=−18±4 mV. Activation time course could be fitted with a single exponential, τd being 1.1 ms close to threshold and 0.5 ms at +10 mV. Deactivation occurred with a similar fast time course.7.Inactivation. Steady-state inactivation was evaluated by stepping from more and more positive holding potentials to +10 mV. The data were fitted according to