Tamotsu Mitsuiye

Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice

We recently reported that mice deficient in the programmed cell death-1 (PD-1) immunoinhibitory coreceptor develop autoimmune dilated cardiomyopathy (DCM), with production of high-titer autoantibodies against a heart-specific, 30-kDa protein. In this study, we purified the 30-kDa protein from heart extract and identified it as cardiac troponin I (cTnI), encoded by a gene in which mutations can cause familial hypertrophic cardiomyopathy (HCM). Administration of monoclonal antibodies to cTnI induced dilatation and dysfunction of hearts in wild-type mice. Monoclonal antibodies to cTnI stained the surface of cardiomyocytes and augmented the voltage-dependent L-type Ca2+ current of normal cardiomyocytes. These findings suggest that antibodies to cTnI induce heart dysfunction and dilatation by chronic stimulation of Ca2+ influx in cardiomyocytes.

Sustained Inward Current During Pacemaker Depolarization in Mammalian Sinoatrial Node Cells

Several time- and voltage-dependent ionic currents have been identified in cardiac pacemaker cells, including Na(+) current, L- and T-type Ca(2+) currents, hyperpolarization-activated cation current, and various types of delayed rectifier K(+) currents. Mathematical models have demonstrated that spontaneous action potentials can be reconstructed by incorporating these currents, but relative contributions of individual currents vary widely between different models. In 1995, the presence of a novel inward current that was activated by depolarization to the potential range of the slow diastolic depolarization in rabbit sinoatrial (SA) node cells was reported. Because the current showed little inactivation during depolarizing pulses, it was called the sustained inward current (I(st)). A similar current is also found in SA node cells of the guinea pig and rat and in subsidiary pacemaker atrioventricular node cells. Recently, single-channel analysis has revealed a nicardipine-sensitive, 13-pS Na(+) current, which is activated by depolarization to the diastolic potential range in guinea pig SA node cells. This channel differs from rapid voltage-gated Na(+) or L-type Ca(2+) channels both in unitary conductance and gating kinetics. Because I(st) was observed only in spontaneously beating SA node cells, ie, it was absent in quiescent cells dissociated from the same SA or atrioventricular node, an important role of I(st) for generation of intrinsic cardiac automaticity was suggested.

The sustained inward current in sino-atrial node cells of guinea-pig heart

Abstract Single myocytes were dissociated from the sino-atrial (SA) node of guinea-pig hearts. Only a quite small fraction of the cell population showed spontaneous action potentials and these cells were characterized by the presence of the hyperpolarization-activated cation current If , the delayed rectifier K+ current IK and the L-type Ca2+ current ICa,L as well as by the absence of both the transient outward current Ito and the inward rectifier K+ current IK,1. After blocking If and IK, depolarizing pulses from –80 mV revealed a large nicardipine-sensitive late current (NSLC). The NSLC was scarcely affected by decreasing extracellular [Ca2+] ([Ca2+]o) from 1.8 to 0.1 mM, while it was decreased significantly by depleting [Na+]o, differently from ICa,L. NSLC was blocked by nicardipine and was increased by Bay K 8644. NSLC was increased by isoprenaline and the additional application of acetylcholine reversed the increase of this current. We conclude that NSLC is largely composed of Ist described in the rabbit SA node pacemaker cells, and that Ist is unique for the pacemaker cells in mammalian SA node cells. Most of the quiescent cells showed neither If nor Ist.

Cell DistensionInduced Increase of the Delayed Rectifier K+ Current in Guinea Pig Ventricular Myocytes

Abstract Single ventricular myocytes of guinea pig heart were distended by applying a positive pressure of 5 to 20 mm Hg in the pipette during the whole-cell voltage clamp. The amplitude of delayed rectifier K + current (I K ) was increased by ≈1.5 times, whereas the inward rectifier K + current was scarcely affected. The increase of I K was reversible by applying a negative pressure of −10 to −30 mm Hg accompanied by shrinkage of the inflated cell. This response of I K was largely attributed to the E-4031–insensitive component of I K . The fully activated current amplitude, measured using long-lasting depolarizing pulses (>30 seconds) to +60 mV, was increased by the cell distension. The activation time course of I K during the long pulse consisted of more than three exponential components, and the slowest time constant was decreased by the distension from control 20.2±7.7 seconds (n=4) to 7.6±1.6 seconds (n=5). We failed to detect an involvement of microtubules or microfilaments, protein kinase C, and Ca 2+ in the inflation-mediated increase of I K .

Nicardipine-sensitive Na+-mediated single channel currents in guinea-pig sinoatrial node pacemaker cells.

1 The Na+‐dependent inward currents underlying slow diastolic depolarization of sinoatrial (SA) node cells were examined. Using a Na+‐rich, Ca2+‐free pipette solution a novel single channel current was recorded in addition to the conventional Na+ and L‐type Ca2+ currents. The current (termed ist, as it reflects the whole‐cell sustained inward current, Ist) does not show obvious inactivation during a 700 ms depolarization and is unique in having a smaller amplitude (1·1 ± 0·18 pA at −60 mV, n= 12) than the Na+ current through conventional Na+ (≈3·3 pA) and Ca2+ channels (9·6 ± 0·32 pA at −60 mV, n= 8). The mean unitary conductance of ist channels was 13·3 pS. 2 The recording of ist was infrequent, was observed only in spontaneously beating SA node cells, and was facilitated by adding Bay‐K 8644 to the pipette solution. Overlapping of ist events was observed and ist was abolished by bath application of nicardipine. 3 In the ensemble average, the activation of ist was evident by depolarization beyond −70 mV, and the dynamic voltage range of activation (‐70 to −30 mV) encompassed the extent of the slow diastolic depolarization. The current density of ist was 0·33 pA pF−1 at −60 mV, as estimated from the number of channels per membrane patch, the open probability and the unitary amplitude. 4 Cumulative histograms for both open and closed times were fitted with a sum of two exponential components. The slow time constants decreased with depolarization, while the fast time constants and the fraction of the fast component were voltage independent. The number of bursts per sweep increased with depolarization. The time constant of the first latency histogram was about two orders of magnitude larger than those in cardiac L‐type Ca2+ channels and decreased with depolarization. 5 It is suggested that the ist channels might be responsible for the whole‐cell Ist.

CHANGES IN CELL VOLUME INDUCED BY ION CHANNEL FLUX IN GUINEA-PIG CARDIAC MYOCYTES

1. The cell width of guinea‐pig ventricular myocytes was measured using an optic device during patch‐clamp experiments and the relationship between the ion channel flux and changes in cell volume was examined.

SARCOMERE LENGTH DURING CONTRACTION OF ISOLATED GUINEA-PIG VENTRICULAR MYOCYTES

Abstract An improved method was developed for measuring sarcomere length (SML) during twitch contractions of single cardiac ventricular myocytes, using a charge-coupled photodiode array self-scanning at a rate of 1.5 ms/element. The average resting SML of 111 cells was 1.88±0.04 µm (mean ±SD). When contractions were triggered by action potentials under perforated-patch conditions, the time course of SML shortening closely followed changes in cell length. A large variation was observed in contraction time course between myocytes, some cells having a phasic component with a duration at 50% shortening (full-width at half-maximum; FWHM) of approximately 40 ms, while others shortened more slowly (FWHM of phasic component @100 ms). FWHM was highly correlated with relaxation half-time, but with neither action potential duration nor resting SML. The kinetics of slowly contracting cells could not be converted to the rapid type by using conditioning trains or applying isoprenaline. The steady-state SML/pCa relation in ventricular myocytes was measured by applying solutions of various pCa immediately after localized punctures of the surface membrane using a focal laser beam. The Hill coefficient, nH, was @4–5 and K1/2@400–500 nM, but there was no evidence of two populations of cells with different Ca2+ sensitivities.

Inactivation of the cardiac Na+ channels in guinea-pig ventricular cells through the open state.

1. The inactivation kinetics of the Na+ current were investigated using the improved oil‐gap voltage clamp method in single ventricular cells of guinea‐pig hearts. 2. Activation of the Na+ current was observed on depolarization more positive than ‐50 mV from a holding potential of ‐100 mV, and inactivation was complete during these depolarizations. The time course of current decay was fitted by a double exponential at potentials between ‐40 and ‐15 mV, and virtually by a single exponential at more positive potentials. The decay time courses examined either by the double‐pulse protocol or the single‐pulse protocol were similar. 3. The double‐pulse protocol clearly revealed a sigmoidal onset of inactivation on depolarization. The initial delay of inactivation decreased with more positive potentials. The time course of double‐pulse inactivation was reconstructed by integrating the Na+ current recorded by a continuous depolarization. 4. These findings are consistent with the hypothesis that the cardiac Na+ channel inactivates exclusively through the open state.

Effects of tilisolol, a nonselective β-adrenergic blocker, on the membrane currents of isolated guinea pig ventricular myocytes

The effects of tilisolol, a nonselective beta-adrenoceptor blocker, on transmembrane ionic currents were studied in single guinea pig ventricular myocytes by using the whole-cell voltage clamp technique. In the absence of beta-adrenergic stimulation, 10 microM tilisolol, a concentration higher than that used in the clinical therapeutic regimen, did not affect the L-type Ca2+ current (ICa), the inwardly rectifying K+ current (IK1), or the delayed rectifying K+ current (IK). In addition, it did not induce currents through the adenosine triphosphate (ATP)-sensitive K+ channels. However, under the nonselective beta-adrenergic stimulation with 1 microM isoproterenol, 1 microM tilisolol almost completely reversed the agonist-induced increase of IK. The increase of ICa by isoproterenol was blocked only by approximately 30% with tilisolol. We concluded that, at therapeutic concentrations (0.01-0.15 microM), tilisolol is a pure beta-adrenoceptor antagonist that has no direct effects on the transmembrane ionic currents of mammalian ventricular myocytes, such as ICa, IK1, or IK. Comparison of the dose-dependent effects of tilisolol on ICa and IK suggested that tilisolol may selectively inhibit catecholamine-induced increase of IK at the therapeutic concentrations. The virtually selective inhibition of IK, leaving ICa intact, may be favorable to prevent the catecholamine-induced arrhythmia without inhibiting contraction.