Ca(2+) -activated K(+) current is essential for maintaining excitability and gene transcription in early embryonic cardiomyocytes.

AIM: Activity of early embryonic cardiomyocytes relies on spontaneous Ca(2+) oscillations that are induced by interplay between sarcoplasmic reticulum (SR) - Ca(2+) release and ion currents of the plasma membrane. In a variety of cell types, Ca(2+) -activated K(+) current (IK(Ca) ) serves as a link between Ca(2+) signals and membrane voltage. This study aimed to determine the role of IK (Ca) in developing cardiomyocytes. METHODS: Ion currents and membrane voltage of embryonic (E9-11) mouse cardiomyocytes were measured by patch clamp; [Ca(2+) ]i signals by confocal microscopy. Transcription of specific genes was measured with RT-qPCR and Ca(2+) -dependent transcriptional activity using NFAT-luciferase assay. Myocyte structure was assessed with antibody labelling and confocal microscopy. RESULTS: E9-11 cardiomyocytes express small conductance (SK) channel subunits SK2 and SK3 and have a functional apamin-sensitive K(+) current, which is also sensitive to changes in cytosolic [Ca(2+) ]i . In spontaneously active cardiomyocytes, inhibition of IK (Ca) changed action and resting potentials, reduced SR Ca(2+) load and suppressed the amplitude and the frequency of spontaneously evoked Ca(2+) oscillations. Apamin caused dose-dependent suppression of NFAT-luciferase reporter activity, induced downregulation of a pattern of genes vital for cardiomyocyte development and triggered changes in the myocyte morphology. CONCLUSION: The results show that apamin-sensitive IK (Ca) is required for maintaining excitability and activity of the developing cardiomyocytes as well as having a fundamental role in promoting Ca(2+) - dependent gene expression.

Endothelin-1 signalling controls early embryonic heart rate in vitro and in vivo.

AIM: Spontaneous activity of embryonic cardiomyocytes originates from sarcoplasmic reticulum (SR) Ca(2+) release during early cardiogenesis. However, the regulation of heart rate during embryonic development is still not clear. The aim of this study was to determine how endothelin-1 (ET-1) affects the heart rate of embryonic mice, as well as the pathway through which it exerts its effects. METHODS: The effects of ET-1 and ET-1 receptor inhibition on cardiac contraction were studied using confocal Ca(2+) imaging of isolated mouse embryonic ventricular cardiomyocytes and ultrasonographic examination of embryonic cardiac contractions in utero. In addition, the amount of ET-1 peptide and ET receptor a (ETa) and b (ETb) mRNA levels were measured during different stages of development of the cardiac muscle. RESULTS: High ET-1 concentration and expression of both ETa and ETb receptors was observed in early cardiac tissue. ET-1 was found to increase the frequency of spontaneous Ca(2+) oscillations in E10.5 embryonic cardiomyocytes in vitro. Non-specific inhibition of ET receptors with tezosentan caused arrhythmia and bradycardia in isolated embryonic cardiomyocytes and in whole embryonic hearts both in vitro (E10.5) and in utero (E12.5). ET-1-mediated stimulation of early heart rate was found to occur via ETb receptors and subsequent inositol trisphosphate receptor activation and increased SR Ca(2+) leak. CONCLUSION: Endothelin-1 is required to maintain a sufficient heart rate, as well as to prevent arrhythmia during early development of the mouse heart. This is achieved through ETb receptor, which stimulates Ca(2+) leak through IP3 receptors.

Activation of Ca(2+)-dependent protein kinase II during repeated contractions in single muscle fibres from mouse is dependent on the frequency of sarcoplasmic reticulum Ca(2+) release.

AIM: To investigate the importance and contribution of calmodulin-dependent protein kinase II (CaMKII) activity on sarcoplasmic reticulum (SR) Ca(2+)-release in response to different work intensities in single, intact muscle fibres. METHODS: CaMKII activity was blocked in single muscle fibres using either the inhibitory peptide AC3-I or the pharmacological inhibitor KN-93. The effect on tetanic force production and [Ca(2+)](i) was determined during work of different intensities. The activity of CaMKII was assessed by mathematical modelling. RESULTS: Using a standard protocol to induce fatigue (50x 70 Hz, 350 ms duration, every 2 s) the number of stimuli needed to induce fatigue was decreased from 47 +/- 3 contractions in control to 33 +/- 3 with AC3-I. KN-93 was a more potent inhibitor, decreasing the number of contractions needed to induce fatigue to 15 +/- 3. Tetanic [Ca(2+)](i) was 100 +/- 11%, 97 +/- 11% and 67 +/- 11% at the end of stimulation in control, AC3-I and KN-93 respectively. A similar inhibition was obtained using a high intensity protocol (20x 70 Hz, 200 ms duration, every 300 ms). However, using a long interval protocol (25x 70 Hz, 350 ms duration, every 5 s) no change was observed in either tetanic [Ca(2+)](i) or force when inhibiting CaMKII. A mathematical model used to investigate the activation pattern of CaMKII suggests that there is a threshold of active CaMKII that has to be surpassed in order for CaMKII to affect SR Ca(2+) release. CONCLUSION: Our results show that CaMKII is crucial for maintaining proper SR Ca(2+) release and that this is regulated in a work intensity manner.

Connexin-mimetic peptide Gap 27 decreases osteoclastic activity.

BACKGROUND: Bone remodelling is dependent on the balance between bone resorbing osteoclasts and bone forming osteoblasts. We have shown previously that osteoclasts contain gap-junctional protein connexin-43 and that a commonly used gap-junctional inhibitor, heptanol, can inhibit osteoclastic bone resorption. Since heptanol may also have some unspecific effect unrelated to gap-junctional inhibition we wanted to test the importance of gap-junctional communication to osteoclasts using a more specific inhibitor. METHODS: A synthetic connexin-mimetic peptide, Gap 27, was used to evaluate the contribution of gap-junctional communication to osteoclastic bone resorption. We utilised the well-characterised pit-formation assay to study the effects of the specific gap-junctional inhibitor to the survival and activity of osteoclasts. RESULTS: Gap 27 caused a remarked decrease in the number of both TRAP-positive mononuclear and multinucleated rat osteoclasts cultured on bovine bone slices. The decrease in the cell survival seemed to be restricted to TRAP-positive cells, whereas the other cells of the culture model seemed unaffected. The activity of the remaining osteoclasts was found to be diminished by measuring the percentage of osteoclasts with actin rings of all TRAP-positive cells. In addition, the resorbed area in the treated cultures was greatly diminished. CONCLUSIONS: On the basis of these results we conclude that gap-junctional communication is essential for the action of bone resorbing osteoclasts and for proper remodelling for bone.

Cardiac mechanotransduction: from sensing to disease and treatment.

In heart muscle a mechanical stimulus is sensed and transformed into adaptive changes in cardiac function by a process called mechanotransduction. Adaptation of heart muscle to mechanical load consists of neurohumoral activation and growth, both of which decrease the initial load. Under prolonged overload this process becomes maladaptive, leading to the development of left ventricular hypertrophy and ultimately to heart failure. Widespread synergism and crosstalk among a variety of molecules and signals involved in hypertrophic signaling pathways make the prevention or treatment of left ventricular hypertrophy and heart failure a challenging task. Therapeutic strategies should include either a complete and continuous reduction of load or normalization of left ventricular mass by interventions aimed at specific targets involved in mechanotransduction.

cAMP- and cGMP-independent stretch-induced changes in the contraction of rat atrium.

The stretch-induced changes in contraction force, cAMP and cGMP in isolated rat left atrium were studied. Increasing the diastolic intra-atrial pressure from 1 cmH2O to 8 cmH2O caused an immediate (<500 ms) increase in contraction force, the magnitude of which was 2.24+/-0.29 (n=6) times the force elicited by 1 cmH2O. This was followed by a slower, gradual increase of the force, which was maximal 8 min after the stretch (4.33+/-0.31, n=6). These phenomena were not accompanied by changes in the cAMP (n=24) or cGMP (n=24) concentrations within the tissue at any duration of stretch tested (2, 8, 20 and 36 min, n=6 at each time point). Furthermore, it was estimated that if the beta-adrenergic receptor agonist isoprenaline (100 nM) was used to produce an increase of the contraction force of the same magnitude as that induced by stretch, the cAMP concentration was greater (4.20+/-0.29 pmol/mg, n=5, P<0.001) when compared to that produced after 20 min of stretch (2.69+/-0.12 pmol/mg, n=6). Even without significantly changing the cGMP concentration, isoprenaline significantly increased the [cAMP]/[cGMP] ratio (3.4+/-0.36, n=5, P<0.01) compared to stretch (1.95+/-0.14, n=6). This result shows that in the rat atrium stretch does not regulate the production or breakdown of cyclic nucleotides (cAMP or cGMP). Thus it seems very unlikely that the effects of stretch on rat atrium function are caused by cAMP or cGMP.

Intracellular acidosis modulates the stretch-induced changes in E-C coupling of the rat atrium.

By inducing a small reduction of the intracellular pH (0.18 units) with 20 mmol L-1 propionate we demonstrated that acidification changed the responses of isolated rat atria to stretch. Stretch (increase of the intra-atrial pressure) in normal pH increased the Ca2+ transients' amplitude (Indo-1 fluorescence) from 0.26 +/- 0.09 in 1 mmHg to 0.36 +/- 0.13 in 4 mmHg (P < 0.05, n=6), without affecting the diastolic [Ca2+]i level (n.s. n=6). The changes in Ca2+ balance during stretch were accompanied by a biphasic increase in the contraction force. Five minutes of continuous stretch increased the action potential duration (APD90%, P < 0.01, n=13) and decreased the APD15% (P < 0.001, n=13). During acidosis, the stretch-induced increase of the Ca2+ transient amplitude (0.4 +/- 0. 13 vs. 0.3 +/- 0.08, P < 0.05, n=6) was accompanied by the increase of the diastolic [Ca2+]i (1.16 +/- 0.07, P < 0.05, n=6) compared with non-acidotic control (1.06 +/- 0.06, n=6). Acidic intracellular pH also inhibited the stretch-induced changes in the action potentials (n=10) and slowed down the development of the contractile force during stretch. The results showed that acidosis modulates the mechanotransduction. It does this by interfering with the intracellular Ca2+ balance, inhibiting the Ca2+ extrusion mechanisms and reducing the Ca2+-buffering power of the cells. The physiological and pathological processes associated with stretch are therefore modulated by intracellular pH owing to its concerted effects on intracellular Ca2+ handling caused by a competitive inhibition of various Ca2+-binding molecules.

Role of the sarcoplasmic reticulum in the modulation of rat cardiac action potential by stretch.

We have investigated the role of sarcoplasmic reticulum (SR) in the modulation on rat action potentials by stretch. The action potentials were recorded intracellularly from rat atrial myocytes in an isolated atrial preparation with small, physiological stretch produced by pressure (1-3 mmHg) inside the atria. The SR function was inhibited by pharmacological interventions, either with ryanodine (100 nmol L-1), thapsigargin (10 nmol L-1) or caffeine (1 mmol L-1). The duration of action potentials was increased by stretch from 1 to 3 mmHg. The repolarization indices APD30% (P < 0.05), APD60% (P < 0.01), and APD90% (P < 0.01) were all increased significantly (n=10). Ryanodine, thapsigargin, and caffeine inhibited this prolongation, or even reversed the effect with repolarization indices APD30% (P < 0.05) and APD60% (P < 0.05) which decreased in stretch with thapsigargin treatment. As a conclusion, we suggest that the SR and the intracellular calcium balance play an important role in the modulation of the shape of the rat atrial action potential during stretch.

Potentiation of stretch-induced atrial natriuretic peptide secretion by intracellular acidosis.

We sought to investigate whether atrial myocyte contraction and secretion of the atrial natriuretic peptide (ANP) are affected in the same manner by intervention in intracellular Ca(2+) handling by acidosis. The effects of propionate (20 mM)-induced intracellular acidosis on the stretch-induced changes in ANP secretion, contraction force, and intracellular Ca(2+) concentration ([Ca(2+)](i)) were studied in the isolated rat atrium. The stretch of the atrium was produced by increasing the intra-atrial pressure of the paced and superfused preparation. Contraction force was estimated from pressure pulses generated by the contraction of the atrium. Intracellular Ca(2+) was measured from indo 1-AM-loaded atria, and ANP was measured by radioimmunoassay from the perfusate samples collected during interventions. Intracellular pH of the atrial myocytes was measured by a fluorescent indicator (BCECF)-based imaging system. Intracellular acidification caused by 20 mM propionic acid (0.18 pH units) potentiated the stretch-induced (intra-atrial pressure from 1 to 4 mmHg) ANP secretion, causing a twofold secretion compared with nonacidotic controls. Simultaneously, the responsiveness of the atrial contraction to stretch was reduced (P < 0.05, n = 7). Stretch augmented the systolic indo 1-AM transients in acidic (P < 0.05, n = 6) and nonacidic atria (P < 0.05, n = 6). However, during acidosis this was accompanied by an increase of the diastolic indo 1-AM ratio (P < 0.05, n = 6). Cooccurrence of stretch and acidosis caused an increase in systolic and diastolic [Ca(2+)](i) and potentiated the stretch-induced ANP secretion, whereas the contraction force and its stretch sensitivity were decreased. This mechanism may be involved in ischemia-induced ANP secretion, suggesting a role for ANP secretion as an indicator of contractile dysfunction.

Mechanisms of stretch-induced changes in [Ca2+]i in rat atrial myocytes: role of increased troponin C affinity and stretch-activated ion channels.

To study the effects of stretch on the function of rat left atrium, we recorded contraction force, calcium transients, and intracellular action potentials (APs) during stretch manipulations. The stretch of the atrium was controlled by intra-atrial pressure. The Frank-Starling behavior of the atrium was manifested as a biphasic increase of the contraction force after increasing the stretch level. The development of the contraction force after step increase of the stretch (intra-atrial pressure from 1 to 3 mm Hg) was accompanied by the increase in the amplitude of the calcium transients (P<0.05, n=4) and decrease in the time constant of the Ca2+ transient decay. The APs of the individual myocytes were also affected by stretch; the duration of the AP was decreased at positive voltages (AP duration at 15% repolarization level, P<0.001; n=13) and increased at negative voltages (AP duration at 90% repolarization level, P<0. 01; n=13). To study the mechanisms causing these changes we developed a mathematical model describing [Ca2+]i and electrical behavior of single rat atrial myocytes. Stretch was simulated in the model by increasing the troponin (TnC) sensitivity and/or applying a stretch-activated (SA) calcium influx. We mimicked the Ca2+ influx by introducing a nonselective cationic conductance, the SA channels, into the membrane. Neither of the 2 plausible mechanosensors (TnC or SA channels) alone could produce similar changes in the Ca2+ transients or APs as seen in the experiments. The model simulated the effects of stretch seen in experiments best when both the TnC affinity and the SA conductance activation were applied simultaneously. The SA channel activation led to gradual augmentation of Ca2+ transients, which modulated the APs through increased Na+/Ca2+-exchanger inward current. The role of TnC affinity change was to modulate the Ca2+ transients, stabilize the diastolic [Ca2+]i, and presumably to produce the immediate increase of the contraction force after stretch seen in experiments. Furthermore, we found that the same mechanism that caused the normal physiological responses to stretch could also generate arrhythmogenic afterpotentials at high stretch levels in the model.

Evidence for cAMP-independent mechanisms mediating the effects of adrenomedullin, a new inotropic peptide.

BACKGROUND: Adrenomedullin (ADM), a new vasorelaxing and natriuretic peptide, may function as an endogenous regulator of cardiac function, because ADM and its binding sites have been found in the heart. We characterize herein the cardiac effects of ADM as well as the underlying signaling pathways in vitro. METHODS AND RESULTS: In isolated perfused, paced rat heart preparation, infusion of ADM at concentrations of 0.1 to 1 nmol/L for 30 minutes induced a dose-dependent, gradual increase in developed tension, whereas proadrenomedullin N-20 (PAMP; 10 to 100 nmol/L), a peptide derived from the same gene as ADM, had no effect. The ADM-induced positive inotropic effect was not altered by a calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP8-37, or H-89, a cAMP-dependent protein kinase inhibitor. ADM also failed to stimulate ventricular cAMP content of the perfused hearts. Ryanodine (3 nmol/L), a sarcoplasmic reticulum Ca2+ release channel opener, suppressed the overall ADM-induced positive inotropic effect. Pretreatment with thapsigargin (30 nmol/L), which inhibits sarcoplasmic reticulum Ca2+ ATPase and depletes intracellular Ca2+ stores, attenuated the early increase in developed tension produced by ADM. In addition, inhibition of protein kinase C by staurosporine (10 nmol/L) and blockade of L-type Ca2+ channels by diltiazem (1 micromol/L) significantly decreased the sustained phase of ADM-induced increase in developed tension. Superfusion of atrial myocytes with ADM (1 nmol/L) in isolated left atrial preparations resulted in a marked prolongation of action potential duration between 10 and -50 mV transmembrane voltage, consistent with an increase in L-type Ca2+ channel current during the plateau. CONCLUSIONS: Our results show that ADM enhances cardiac contractility via cAMP-independent mechanisms including Ca2+ release from intracellular ryanodine- and thapsigargin-sensitive Ca2+ stores, activation of protein kinase C, and Ca2+ influx through L-type Ca2+ channels.

Effect of gadolinium on stretch-induced changes in contraction and intracellularly recorded action- and afterpotentials of rat isolated atrium.

1. Atrial arrhythmias, like atrial fibrillation and extrasystoles, are common in clinical situations when atrial pressure is increased. Although cardiac mechanoelectrical feedback has been under intensive study for many years, the mechanisms of stretch-induced arrhythmias are not known in detail. This is partly due to methodological difficulties in recording intracellular voltage during stretch stimulation. In this study we investigated the effects of gadolinium (Gd3+), a blocker of stretch-activated (SA) channels, on stretch-induced changes in rat atrial action potentials and contraction force. 2. By intracellular voltage recordings from rat isolated atria we studied the effects of Gd3+ (80 microM) on stretch-induced changes in action potentials. The stretch was induced by increasing pressure inside the atrium (1 mmHg to 7 mmHg). An elastic electrode holder that moved along the atrial tissue was used in the recordings. Thus the mechanical artifacts were eliminated and the cell-electrode contact was made more stable. To examine the influence of Gd3+ on atrial contraction we stretched the atria at different diastolic pressure levels (1 to 7 mmHg) with Gd3+ application of (80 microM) or diltiazem (5.0 microM). Contraction force was monitored by recording the pressure changes generated by the atrial contractions. 3. Our results show that: (1) atrial stretch induces delayed afterdepolarizations (DADs), increase in action potential amplitude and increase in relative conduction speed; (ii) Gd3+ blocks stretch-induced DADs and action potential changes; (iii) Gd3+ inhibits pressure-stimulated increase in the atrial contraction force, while similar inhibition is not observed with diltiazem, a blocker of L-type calcium channels. 4. This study suggests that Gd3+ inhibits stretch-induced changes in cell electrophysiology and contraction in the rat atrial cells and that the effects of gadolinium are due to rather specific block of stretch-activated ion channels with only a small effect on voltage-activated calcium channels.