Guy Vassort

PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle: defective regulation in heart failure

The type 1 ryanodine receptor (RyR1) on the sarcoplasmic reticulum (SR) is the major calcium (Ca2+) release channel required for skeletal muscle excitation–contraction (EC) coupling. RyR1 function is modulated by proteins that bind to its large cytoplasmic scaffold domain, including the FK506 binding protein (FKBP12) and PKA. PKA is activated during sympathetic nervous system (SNS) stimulation. We show that PKA phosphorylation of RyR1 at Ser2843 activates the channel by releasing FKBP12. When FKB12 is bound to RyR1, it inhibits the channel by stabilizing its closed state. RyR1 in skeletal muscle from animals with heart failure (HF), a chronic hyperadrenergic state, were PKA hyperphosphorylated, depleted of FKBP12, and exhibited increased activity, suggesting that the channels are “leaky.” RyR1 PKA hyperphosphorylation correlated with impaired SR Ca2+ release and early fatigue in HF skeletal muscle. These findings identify a novel mechanism that regulates RyR1 function via PKA phosphorylation in response to SNS stimulation. PKA hyperphosphorylation of RyR1 may contribute to impaired skeletal muscle function in HF, suggesting that a generalized EC coupling myopathy may play a role in HF.

Calcium current in single cells isolated from normal and hypertrophied rat heart. Effects of beta-adrenergic stimulation.

The L-type calcium current was investigated in normal and hypertrophied rat ventricular myocytes as a possible cause of the action potential lengthening that has been reported during hypertrophy. Regulation of the calcium current (ICa) by a beta-adrenergic agonist (isoproterenol) was also analyzed since beta-agonist-induced positive inotropy is less marked in hypertrophied heart. Left ventricular hypertrophy was induced by stenosis of the abdominal aorta. For recording ICa, the whole-cell patch-clamp technique was used. Potassium currents were suppressed by replacing K+ ions with Cs+ ions in both the extracellular and intracellular media, and sodium current was blocked by 50 microM tetrodotoxin. The Ca2+ current was larger in hypertrophied cells (2.2 +/- 0.6 nA [n= 31]) than in normal cells (1.2 +/- 0.5 nA [n = 33]). However, if one relates ICa amplitude to the cell membrane area, as estimated by membrane capacitance measurement, no significant difference was observed in current density (8.5 +/- 2.5 pA/pF [n = 31] and 8.3 +/- 2.1 pA/pF [n = 33] in hypertrophied and in normal cells, respectively). In both cell types, ICa displayed the same voltage and time dependence. When expressed as a percentage, the maximal increase in ICa amplitude that was obtained with 100 nM isoproterenol was less in hypertrophied cells (+78%) than in normal cells (+120%). The sensitivity of ICa to beta-adrenergic stimulation was not modified: EC50 was 3.8 nM for hypertrophied cells and 4.8 nM for normal cells. Forskolin and cyclic AMP were as effective in both cell types. Stimulation of ICa by beta-adrenergic agonist was decreased in agreement with a reduced number of binding sites of beta-agonists and/or an altered coupling of the G-proteins.

Alpha 1‐adrenergic effects on intracellular pH and calcium and on myofilaments in single rat cardiac cells.

1. The cellular effects of alpha 1‐adrenoceptor stimulation by phenylephrine were studied in the presence of propranolol in single cells isolated from the ventricles of rat hearts. 2. Phenylephrine (10‐100 microM) induced a biphasic pattern of inotropism in these cells: a transient negative followed by a sustained positive inotropic effect as usually observed in cardiac tissues. 3. In Snarf‐1‐loaded cells, phenylephrine induced an alkalinization. This effect was reversible on wash‐out and inhibited by prazosin, an alpha 1‐adrenoceptor antagonist. 4. The alpha 1‐adrenoceptor‐mediated increase in intracellular pH (pHi) was 0.1 pH unit in HEPES buffer containing 4.4 mM‐NaHCO3 and in Krebs buffer containing 25 mM‐NaHCO3. 5. The alkalinization was blocked by the Na(+)‐H+ antiport blocker, ethylisopropylamiloride (EIPA). 6. The recovery from an acidosis induced by a NH4Cl pre‐pulse was accelerated by phenylephrine. The phenylephrine‐induced alkalinization was attributed to activation of the Na(+)‐H+ antiport. 7. Despite its ability to increase pHi, phenylephrine did not alter Ca2+ current amplitude and kinetics. 8. Ca2+ transients recorded in Indo‐1‐loaded cells were not augmented by phenylephrine. Diastolic calcium level was decreased. 9. In single skinned cells, the Ca2+ sensitivity of the contractile proteins was increased by a pre‐treatment with phenylephrine even when the alpha 1‐adrenoceptor‐mediated alkalinizing effect had been prevented by EIPA. 10. These results lead us to propose that the alpha 1‐adrenergic‐induced positive inotropic response of heart muscle could result from an increased sensitivity of the myofilaments to Ca2+ ions. This alpha 1‐adrenoceptor‐mediated Ca2+ sensitization could result both from an intracellular alkalinization and from a direct effect on contractile proteins.

Membrane potential and slow inward current dependence of frog cardiac mechanical activity

SummaryBoth contractile response and ionic currents are recorded during voltage clamp experiments on frog atrial trabecles.In Ringer solution, tension elicited by depolarizing steps of different duration and amplitude may be considered as composed of two elements.One depends on the slow inward current; the estimated variation in [Ca++]i can account for the tension elicited. At its level, the competition between Ca and Na ions is manifest.The other component, depending on the membrane potential, exists even in absence of external Ca ions. Such a component of mechanical response suggests an intracellular source of activator-calcium and these ions can be displaced by membrane potential. Moreover, both this component and the absence of threshold in the tension development agree with the fact that the membrane potential controls the diastolic tension.These two components share in the contraction elicited by action potential.

Inositol phosphate production following α1-adrenergic, muscarinic or electrical stimulation in isolated rat heart

A possible participation of polyphosphoinositide metabolism in the excitation‐contraction coupling in heart was investigated. Isolated rat ventricles prelabelled with myo‐[2‐3H]inositol were stimulated by conditions that increase mechanical activity. Both noradrenaline and carbachol increased the basal level of IP3 IP2 and IP by the activation of α1‐adrenergic and muscarinic receptors, respectively. Electrical stimulation accelerated inositol lipid degradation by phospholipase C thus enhancing the IP3 level as compared to quiescent ventricles. It is proposed that IP3 may be involved in excitation‐contraction coupling in cardiac tissue.

Neurohormonal control of calcium sensitivity of myofilaments in rat single heart cells.

To investigate the changes in the properties of cardiac contractile proteins due to neurohormonal stimulation, different agonists were applied to single cells isolated from rat ventricle. Cells were then rapidly skinned by Triton X-100, and force was recorded after gluing the cells to a strain gauge. The skinned cells had mechanical properties very similar to those described for thin trabeculas. Tension-pCa relations were highly reproducible from one cell to another, with sarcomere length fixed at 2.1 microns. The application of alpha 1-adrenergic and muscarinic agonists, which increase the turnover of phosphatidylinositol, for 5 minutes before skinning the cells increased the sensitivity of the myofilaments to calcium, as indicated by a leftward shift of the tension-pCa relation, whereas beta-adrenergic stimulation induced a rightward shift. The increase in calcium sensitivity was also evoked by protein kinase C activators such as 1,2-dioctanoylglycerol and phorbol 12-myristate 13-acetate but not by protein kinase C itself or by purinergic agonists, although the latter also increased the turnover of phosphatidylinositol. Incubation of the skinned cells with phosphatase reversed the alterations in calcium sensitivity induced by previous agonist stimulation of the intact cells. In conclusion, this study demonstrates a potentially influential mechanism for the physiological regulation of cardiac muscle contractility.

Effects of aldosterone on transient outward K+ current density in rat ventricular myocytes.

1 Aldosterone, a major ionic homeostasis regulator, might also regulate cardiac ion currents. Using the whole‐cell patch‐clamp technique, we investigated whether aldosterone affects the 4‐aminopyridine‐sensitive transient outward K+ current (Ito1). 2 Exposure to 100 nm aldosterone for 48 h at 37 °C produced a 1.6‐fold decrease in the Ito1 density compared to control myocytes incubated without aldosterone. Neither the time‐ nor voltage‐dependent properties of the current were significantly altered after aldosterone treatment. RU28318 (1 μm), a specific mineralocorticoid receptor antagonist, prevented the aldosterone‐induced decrease in Ito1 density. 3 When myocytes were incubated for 24 h with aldosterone, concentrations up to 1 μm did not change Ito1 density, whereas L‐type Ca2+ current (ICa,L) density increased. After 48 h, aldosterone caused a further increase in ICa,L. The delay in the Ito1 response to aldosterone might indicate that it occurs secondary to an increase in ICa,L. 4 After 24 h of aldosterone pretreatment, further co‐incubation for 24 h either with an ICa,L antagonist (100 nm nifedipine) or with a permeant Ca2+ chelator (10 μm BAPTA‐AM) prevented a decrease in Ito1 density. 5 After 48 h of aldosterone treatment, we observed a 2.5‐fold increase in the occurrence of spontaneous Ca2+ sparks, which was blunted by co‐treatment with nifedipine. 6 We conclude that aldosterone decreases Ito1 density. We suggest that this decrease is secondary to the modulation of intracellular Ca2+ signalling, which probably arises from the aldosterone‐induced increase in ICa,L. These results provide new insights into how cardiac ionic currents are modulated by hormones.

Cardiac T-type calcium current: pharmacology and roles in cardiac tissues.

Modulation of T‐Type Calcium Channels. A low threshold, voltage‐gated calcium current is reported in most cardiac tissues but rarely in ventricular cells. This article reports some recently described characteristics and discusses their possible pathophysiologic implications. It also reviews the alterations induced in this current by a variety of chemical agents including several neuromediators in cardiac and other tissues.

Ionic basis of ventricular arrhythmias in remodeled rat heart during long-term myocardial infarction

OBJECTIVE Deleterious electrical abnormalities evolve during myocardial infarction. The goal of this study was to analyse current changes during the late decompensated phase of heart disease induced by coronary ligation and to compare them in various heart regions. METHODS Young rats were submitted to left coronary ligature. After 4-6 months, cells were enzymatically dissociated and isolated from the upper part basal region of the left ventricle, as well as from the septum, apex and the right ventricle before being studied under whole-cell patch-clamp. RESULTS Basal L-type Ca2+ current, ICaL elicited at +10 mV did not exhibit regional dependence neither in control nor after post-myocardial infarction (PMI). ICaL showed both a significantly reduced peak amplitude (17.1 +/- 2.8 pA/pF versus 9.9 +/- 1.4 pA/pF in seven control and seven PMI hearts, n = 32 and 40, respectively) and a slower inactivation, such that the amount of inward charges during a 200 ms-depolarizing pulse was nearly unchanged. beta-Adrenergic stimulation was less effective in increasing ICaL in PMI cells but it slowed inactivation further. Significant differences in the K+ currents were observed. A regional distribution was seen for Ito only, with the largest amplitude in the right ventricle (in pA/pF: 23.1 +/- 2.4, 18.2 +/- 3.9, 14.8 +/- 2.4, 8.3 +/- 1.7 in the right ventricle, apex, septum and left ventricle, respectively n = 8, 7, 8 and 9). This was also true in failing heart cells despite Ito being halved in each of the four regions (in pA/pF: 12.2 +/- 2.5, 11.2 +/- 1.9, 5.1 +/- 1.0 and 4.8 +/- 1.0, respectively n = 12, 12, 11 and 13). IK1 was also significantly reduced by 20% in the PMI cells. Two-way analyses of variance demonstrated the absence of interaction between the topographical origin of the cells and the physiological state of the rats. The alpha 1-adrenergic agonist, methoxamine significantly reduced Ito and IK1 to the same extent in both sham and PMI cells, by about 35% and 20% respectively. CONCLUSIONS Long-term left coronary occlusion induces significant alterations in both Ca2+ and K+ currents that occur with similar amplitude in both ventricles. They include a marked reduction in Ito amplitude as well as a slowing of ICaL inactivation. Both factors could contribute to the disturbances in cellular electrical behaviour and the occurrence of arrhythmias in the post-myocardial infarcted heart.

Simultaneous activation of p38 MAPK and p42/44 MAPK by ATP stimulates the K+ current ITREK in cardiomyocytes.

Living cells exhibit multiple K+ channel proteins; among these is the recently reported atypical two-pore domain K+ channel protein TREK-1. Most K+ currents are modulated by neurohormones and under various pathological conditions. Here, in rat ventricular cardiomyocytes using the whole-cell patch-clamp technique, we characterize for the first time a native TREK-1-like current (ITREK) that is activated by ATP, a purine agonist applied at a micromolar range. This current is sensitive to arachidonic acid, intracellular acidosis, and various K+ current inhibitors. Reverse transcription-polymerase chain reaction reveals the presence of a TREK-1-like mRNA in rat cardiomyocytes that shows 93% identity with mouse TREK-1. ATP effects are greatly attenuated in the presence of arachidonic acid or HCO− 3-induced intracellular acidosis. Using a series of inhibitors, we further demonstrate that the ATP-induced stimulation of ITREKimplies the activation of cytosolic phospholipase A2 and the release of arachidonic acid. These events require the simultaneous involvement of p38 MAPK and p42/44 MAPK, respectively, via a cAMP-dependent protein kinase and a tyrosine kinase pathway, whereas the two MAPKs conjugate to activate a mitogen- and stress-activated protein kinase (MSK-1). Our results thus demonstrate the occurrence of a TREK-1-like current in cardiac cells whose activation by purine agonists implies a dual-MAPK cytosolic pathway.