Cyclic mechanical strain causes cAMP-response element binding protein activation by different pathways in cardiac fibroblasts.

The transcription factor cAMP-response element binding protein (CREB) mediates the mechanical strain-induced gene expression in the heart. This study investigated which signaling pathways are involved in the straininduced CREB activation using cultured ventricular fibroblasts from adult rat hearts. CREB phosphorylation was analyzed by immunocytochemistry and ELISA. Cyclic mechanical strain (1 Hz and 5% elongation) for 15 min induced CREB phosphorylation in all CREB-positive fibroblasts. Several signaling transduction pathways can contribute to strain-induced CREB activation. The inhibition of PKA, PKC, MEK, p38-MAPK or PI3-kinase partially reduced the strain-induced CREB phosphorylation. Activation of PKA by forskolin or PKC by PMA resulted in a level of CREB phosphorylation comparable to the reduced level of the strain-induced CREB phosphorylation in the presence of PKA or PKC inhibitors. Signaling pathways involving PKC, MEK, p38-MAPK or PI3-kinase seem to converge during strain-induced CREB activation. PKA interacted additively with the investigated signaling pathways. The strain-induced c-Fos expression can be reduced by PKC inhibition but not by PKA inhibition. Our results suggest that the complete strain-induced CREB phosphorylation involves several signaling pathways that have a synergistic effect. The influence on gene expression is dependent on the level and the time of CREB stimulation. These wide-ranging possibilities of CREB activation provide a graduated control system.

Cyclical mechanical stretch modulates expression of collagen I and collagen III by PKC and tyrosine kinase in cardiac fibroblasts.

Mechanical load and chemical factors as stimuli for the different pattern of the extracellular matrix (ECM) could be responsible for cardiac dysfunction. Since fibroblasts can both synthesize and degrade ECM, ventricular fibroblasts from adult rat hearts underwent cyclical mechanical stretch (CMS; 0.33 Hz) by three different elongations (3%, 6%, 9%) and four different serum concentrations (0%, 0.5%, 5%, 10%) within 24 h. Expression of collagen I and III, as well as matrix metalloproteinase-2 (MMP-2), tissue inhibitor of MMP-2 (TIMP-2), and colligin were analyzed by RNase protection assay. In the absence of serum, 9% CMS increased the mRNA of collagen I by 1.70-fold and collagen III by 1.64-fold. This increase was prevented by the inhibition either of PKC or of tyrosine kinase but not of PKA. Inhibition of PKC or tyrosine kinase itself reduced the expression of collagen I and collagen III mRNA. The mRNA of MMP-2, TIMP-2, and colligin showed the same tendency by stretch. Combined with 10% serum, 6% CMS reduced the mRNA of collagen I (0.62-fold) and collagen III (0.79-fold). Inhibition of PKC or tyrosine kinase, but not of PKA, prevented the reduction of collagen I and collagen III mRNA in 10% serum. The results show that the response of fibroblasts to CMS depends on the serum concentration. At least two signaling pathways are involved in the stretch-induced ECM regulation. Myocardial fibrosis due to ECM remodeling contributes to the dysfunction of the failing heart, which might be attributed to changes in hemodynamic loading.

The stretch-activated potassium channel TREK-1 in rat cardiac ventricular muscle.

OBJECTIVE: The biophysical properties and the regulation of the two-pore-domain potassium channel TREK-1 were studied in rat cardiomyocytes. METHODS: RT-PCR, immunohistochemistry and patch-clamp recording were performed in isolated rat ventricular cardiomyocytes. In some whole-cell-clamp experiments the myocytes were mechanically stretched using a glass stylus. RESULTS: We found strong expression of a splice variant of TREK-1 in rat heart. Immunohistochemistry with antibodies against TREK-1 showed localization of the channel in longitudinal stripes at the external surface membrane of cardiomyocytes. When the cardiomyocytes were mechanically stretched, an outwardly rectifying K+ current component could be detected in whole-cell recordings. In single-channel recordings with symmetrical high K+ solution, two TREK-like channels with 'flickery-burst' kinetics were found: a 'large conductance' K+ channel (132+/-5 pS at positive potentials) and a novel 'low-conductance' channel (41+/-5 pS at positive potentials). The low-conductance channel could be activated by negative pressure in inside-out patches, positive pressure in outside-out patches, intracellular acidification and application of arachidonic acid. Its open probability was strongly increased by depolarization, due to decreased duration of gaps between bursts. The biophysical properties of the two cardiac TREK-like channels were similar to those of TREK-1 channels expressed in HEK293 cells, which both displayed low- and high-conductance modes. CONCLUSIONS: Our results suggest that the two TREK-like channels found in rat cardiomyocytes may reflect two different operating modes of TREK-1. The novel low-conductance channels described here may represent the major operating mode of TREK-1. The current flowing through mechanogated TREK-1 channels may serve to counterbalance the inward current flowing through stretch-activated non-selective cation channels during the filling phase of the cardiac cycle and thus to prevent the occurrence of ventricular extrasystoles.

Organization of Ca2+ release units in excitable smooth muscle of the guinea-pig urinary bladder.

Ca(2+) release from internal stores (sarcoplasmic reticulum or SR) in smooth muscles is initiated either via pharmaco-mechanical coupling due to the action of an agonist and involving IP3 receptors, or via excitation-contraction coupling, mostly involving L-type calcium channels in the plasmalemma (DHPRs), and ryanodine receptors (RyRs), or Ca(2+) release channels of the SR. This work focuses attention on the structural basis for the coupling between DHPRs and RyRs in phasic smooth muscle cells of the guinea-pig urinary bladder. Immunolabeling shows that two proteins of the SR: calsequestrin and the RyR, and one protein the plasmalemma, the L-type channel or DHPR, are colocalized with each other within numerous, peripherally located sites located within the caveolar domains. Electron microscopy images from thin sections and freeze-fracture replicas identify feet in small peripherally located SR vesicles containing calsequestrin and distinctive large particles clustered within small membrane areas. Both feet and particle clusters are located within caveolar domains. Correspondence between the location of feet and particle clusters and of RyR- and DHPR-positive foci allows the conclusion that calsequestrin, RyRs, and L-type Ca(2+) channels are associated with peripheral couplings, or Ca(2+) release units, constituting the key machinery involved in excitation-contraction coupling. Structural analogies between smooth and cardiac muscle excitation-contraction coupling complexes suggest a common basic mechanism of action.

Contraction augments L-type Ca2+ currents in adherent guinea-pig cardiomyocytes.

As integrins are thought to function as mechanoreceptors, we studied whether they could mediate mechanical modulation of the L-type Ca2+ channel current (ICa) in guinea-pig cardiac ventricular myocytes (CVMs). CVMs were voltage clamped with 280 ms pulses from -45 to 0 mV at 0.5 Hz (1.8 mM [Ca2+]o, 22 degrees C). Five minutes after whole-cell access (designated as 0 min) peak ICa was determined from a current-voltage (I-V) curve. Additional recordings were made after 5, 10 and 15 min. At control, ICa was not stable, but ran down during these periods. This run-down of ICa was attenuated by soluble fibronectin (FN) and was changed to an enhancement of ICa when CVMs were attached to FN-coated coverslips. Soluble peptide containing the integrin binding sequence of FN, Arg-Gly-Asp (RGD motif), did not modulate ICa; however, ICa increased in stimulated CVMs attached to RGD peptide-coated coverslips. The effect was not specific to integrins, because attachment to poly-D-lysine-coated coverslips also augmented ICa in stimulated CVMs. Augmentation of ICa by immobilized FN required rhythmical contraction of attached CVMs, because it was attenuated without electrical stimulation and after cell dialysis with the calcium chelator BAPTA. Furthermore, contraction-induced augmentation of ICa in FN-attached CVMs was sensitive to inhibition of protein kinase C (PKC; by Ro-31-8220), inhibition of tyrosine kinase activity (herbimycin A) and cytoskeletal depolymerization (cytochalasin D or colchicine). We attribute augmentation of ICa to the activation of signalling cascades by shear forces that are generated when CVMs contract against attachment; in vivo similar signals may occur when CVMs contract against attachment of integrins to the extracellular matrix.

Altered calcium handling is critically involved in the cardiotoxic effects of chronic beta-adrenergic stimulation.

BACKGROUND: Chronic adrenergic stimulation leads to cardiac hypertrophy and heart failure in experimental models and contributes to the progression of heart failure in humans. The pathways mediating the detrimental effects of chronic beta-adrenergic stimulation are only partly understood. We investigated whether genetic modification of calcium handling through deletion of phospholamban in mice would affect the development of heart failure in mice with transgenic overexpression of the beta1-adrenergic receptor. METHODS AND RESULTS: We crossed beta1-adrenergic receptor transgenic (beta1TG) mice with mice homozygous for a targeted deletion of the phospholamban gene (PLB-/-). Phospholamban ablation dramatically enhanced survival of beta1TG mice. The decrease of left ventricular contractility typically observed in beta1TG mice was reverted back to normal by phospholamban ablation. Cardiac hypertrophy and fibrosis were significantly inhibited in beta1TG/PLB-/- mice compared with beta1TG mice, and the heart failure-specific gene expression pattern was normalized. Analysis of intracellular calcium transients revealed increased diastolic calcium levels and decreased rate constants of diastolic calcium decline in beta1TG mice. In beta1TG/PLB-/- mice, diastolic calcium concentration was normal and rate constants of diastolic calcium decline were greater than in wild-type mice. CONCLUSIONS: We conclude that modification of abnormal calcium handling in beta1TG mice through ablation of phospholamban resulted in a rescue of functional, morphological, and molecular characteristics of heart failure in beta1-adrenergic receptor-transgenic mice. These results imply altered calcium handling as critical for the detrimental effects of beta1-adrenergic signaling.

Na(+)-Ca(2+) exchanger overexpression predisposes to reactive oxygen species-induced injury.

OBJECTIVE: In heart failure (HF), the generation of reactive oxygen species (ROS) is enhanced. It was shown that failing cardiac myocytes are more susceptible to ROS-induced damage, possibly due to increased expression of the sarcolemmal Na-Ca exchanger (NCX). METHODS: We investigated the consequences of increased expression levels of NCX in adult rabbit ventricular cardiomyocytes (via adenovirus-mediated gene transfer, Ad-NCX1-GFP) with respect to tolerance towards ROS. After 48-h incubation, cells were monitored for morphological changes on an inverted microscope. ROS were generated via hydrogen peroxide (H(2)O(2)) (100 micromol/l) and Fe(3+)/nitrilotriacetate (Fe(3+)/NTA, 100/200 micromol/l) for 4 min and cell morphology was followed over 30 min. [Na(+)](i) and [Ca(2+)](i) in native cells were measured using SBFI-AM and Indo1-AM, respectively. RESULTS: In native myocytes, exposure to ROS induced hypercontracture. This was accompanied by a 1.3-fold increase in diastolic Indo1 fluorescence ratio (P<0.05). Overexpression of NCX significantly enhanced development of hypercontracture. After 15 min, the percentage of cells that had undergone hypercontracture (F(hyper)) was 85+/-4% vs. only 44+/-10% in control cells (P<0.05). Inhibition of NCX-mediated Ca(2+) entry with KB-R7943 (5 micromol/l) reduced F(hyper) to 33+/-11% (P<0.05). [Na(+)](i) was increased 2.9-fold 1 min prior to hypercontracture (P<0.05). CONCLUSIONS: ROS-induced hypercontracture is due to Ca(2+) entry via NCX which could be triggered by a concomitant substantial increase in [Na(+)](i). Elevated NCX levels predispose to ROS-induced injury, a mechanism likely contributing to myocyte dysfunction and death in heart failure.

Ion selectivity of stretch-activated cation currents in mouse ventricular myocytes.

Stretch-activated non-selective cation currents ( I(SAC)) constitute a mechanism that can induce cardiac arrhythmias. We studied I(SAC) in mouse ventricular myocytes by stretching part of the cell surface between the patch-pipette and a motor-driven glass stylus. In non-clamped cells, local stretch depolarised and induced after-depolarisations and extrasystoles. In voltage-clamped cells (K(+) currents suppressed) I(SAC) activated by local stretch had a nearly linear voltage dependence and reversed polarity between -12 and 0 mV. Conductance G(SAC) increased with the extent of local stretch. I(SAC) was not a Cl(-) current (insensitivity to replacement of Cl(-) by aspartate(-)). I(SAC) was not a Ca(2+)-activated current (insensitivity to 5 mM intracellular BAPTA). G(SAC) was blocked by 5 micro M GdCl(3) or by 75 mM extracellular (e.c.) CaCl(2). Removal of e.c. CaCl(2) increased G(SAC) 2.5-fold, as if G(SAC) were sensitive to Ca(2+) and Gd(3+). Replacement of 150 mM e.c. Na(+) by 150 mM Cs(+), Li(+), tetraethylammonium (TEA(+)) or N-methyl d-glucosamine (NMDG(+)) yielded currents that suggested for the conductance a selectivity G(Cs)> G(Na)> G(Li)> G(TEA)> G(NMDG). I(SAC) was suppressed by cytochalasin D, as if an intact F-actin cytoskeleton were necessary for activation of I(SAC).

Differential effects of stretch and compression on membrane currents and [Na+]c in ventricular myocytes.

Mechano-electrical feedback was studied in the single ventricular myocytes. A small fraction (approximately 10%) of the cell surface could be stretched or compressed by a glass stylus. Stretch depolarised, shortened the action potential and induced extra systoles. Stretch activated non-selective cation currents (I(ns)) showed a linear voltage dependence, a reversal potential of 0 mV, a pure cation selectivity, and were blocked by 8 microM Gd(3+) or 30 microM streptomycin. Stretch reduced Ca(2+) and K(+) (I(K)) currents. Local compression of broadwise attached cells activated I(K) but not I(ns). Cytochalasin D or colchicin, thought to disrupt the cytoskeleton, suppressed the mechanosensitivity of I(ns) and I(K). During stretch, the cytosolic sodium concentration increased with spatial heterogeneities, local hotspots with [Na(+)](c)>24 mM appeared close to surface membrane and t-tubules (pseudoratiometric imaging using Sodium Green fluorescence). Electronprobe microanalysis confirmed this result and indicated that stretch increased total sodium [Na] in cell compartments such as mitochondria, nuclear envelope and nucleus. Our results obtained by local stretch differ from those obtained by end-to-end stretch (literature). We speculate that channels may be activated not only by axial but also by shear stress, and, that stretch can activate channels outside the deformed sarcomeres via second messenger.

Cardiac fibroblasts and the mechano-electric feedback mechanism in healthy and diseased hearts.

Cardiac arrhythmia is a serious clinical condition, which is frequently associated with abnormalities of mechanical loading and changes in wall tension of the heart. Recent novel findings suggest that fibroblasts may function as mechano-electric transducers in healthy and diseased hearts. Cardiac fibroblasts are electrically non-excitable cells that respond to spontaneous contractions of the myocardium with rhythmical changes of their resting membrane potential. This phenomenon is referred to as mechanically induced potential (MIP) and has been implicated in the mechano-electric feedback mechanism of the heart. Mechano-electric feedback is thought to adjust the frequency of spontaneous myocardial contractions to changes in wall tension, which may result from variable filling pressure. Electrophysiological recordings of single atrial fibroblasts indicate that mechanical compression of the cells may activate a non-selective cation conductance leading to depolarisation of the membrane potential. Reduced amplitudes of MIPs due to pharmacological disruption of F-actin and tubulin suggest a role for the cytoskeleton in the mechano-electric signal transduction process. Enhanced sensitivity of the membrane potential of the fibroblasts to mechanical stretch after myocardial infarction correlates with depression of heart rates. It is assumed that altered electrical function of cardiac fibroblasts may contribute to the increased risk of post-infarct arrhythmia.

Activation and inactivation of a non-selective cation conductance by local mechanical deformation of acutely isolated cardiac fibroblasts.

OBJECTIVE: We describe mechanically induced non-selective cation currents in isolated rat atrial fibroblasts, which might play a role as a substrate for mechano-electrical feedback in the heart. METHODS: Isolated fibroblasts were used for voltage-clamp analysis of ionic currents generating mechanically-induced potentials. Fibroblasts were mechanically deformed (compressed or stretched) by two patch-pipettes. RESULTS: These cells had a resting potential (E(0)) of -37+/-3 mV and an input resistance of 514+/-11 M(Omega). At intracellular pCa 7 (patch-pipette solution), compression of 2 or 3 microm shifted E(0) from -36+/-7 to -17+/-3 mV, and to -10+/-2 mV. Compression by 2 or 3 microm induced a negative difference current (at -45 mV -0.06+/-0.02 and -0.20+/-0.04 nA, respectively) with a reversal potential (E(rev)) of approx. 0 mV. The currents were carried by Na(+), K(+) and Cs(+) ions, and were blocked by application of 8 microM Gd(3+). Stretch of 2 or 3 microm hyperpolarized E(0) from -34+/-4 to -45+/-5, and to -61+/-7 mV and induced a positive difference current (at -45 mV: 0.04+/-0.02 and 0.18+/-0.03 nA) with an E(rev) close to 0 mV. Application of Gd(3+) shifted E(0) to potentials as negative as E(K) (-90+/-4 mV). Cell dialysis with 5 mM BAPTA (pCa 8) or 5 mM Ca(2+)/EGTA (pCa 6) had no influence on non-selective cation currents suggesting that Ca(2+) dependent conductances are unlikely to contribute. CONCLUSION: Compression of the isolated cardiac fibroblast caused depolarization of the membrane by activating inward currents through a non-selective cation conductance (G(ns)). Stretch hyperpolarizes the fibroblast, however, not by Ca(2+) activation of K(+)-conductance. Ion selectivity, E(rev,) and Gd(3+)-sensitivity of stretch suppressed currents suggest that stretch reduces G(ns) that is activated by compression.

Monitoring of ANP secretion from single atrial myocytes using densitometry.

Atrial myocytes secrete atrial natriuretic peptide (ANP) in response to mechanical stretch and can serve as a challenging model for studying stretch-secretion coupling. We have developed a technique for monitoring ANP secretion from single atrial myocytes, using neutral red and a CCD video camera. Atrial-specific granules (ASGs) containing ANP were stained with neutral red. The cells were illuminated with monochromatic light (550 mm) and the grey value monitored within the region of interest (ROI) surrounding the region in which ASGs were densely located. Assuming that neutral red is evenly distributed in ASGs, the change in optical density (OD) was considered to represent the total amount of secretion. Under control, non-stimulated conditions, the OD decreased spontaneously (19.7+/-1.4%/10 min, n=14). Direct mechanical stretch (cell length increased by 20%) with two micropipettes or hypotonic swelling (200 mOsm) accelerated the decrease in OD significantly (48.7+/-7.4%/10 min; n=3, 47.2+/-2.4%/10 min; n=7, respectively). In conclusion, this method allows monitoring of ANP secretion with a relatively high time resolution while mechanical stress is applied. Furthermore, patch-clamp or intracellular perfusion techniques can be combined with the present technique for studying cellular mechanisms of stretch-secretion coupling.

A possible role for atrial fibroblasts in postinfarction bradycardia.

Atrial fibroblasts are considered to modulate the contractile activity of the heart in response to mechanical stretch. In this study we examined whether atrial fibroblasts are possibly involved in bradyarrhythmia, which is a severe complication after myocardial infarction. For this purpose, transmembrane electrical potentials were recorded in cardiac fibroblasts near the sinoatrial node from sham-operated rats and from rats with myocardial infarction. Twenty days after infarction due to coronary artery ligation, the right atrial tissue weights and the sensitivity of the fibroblast membrane potential to mechanical stretch correlated positively with the infarct size. Cardiac growth was enhanced, but the stretch sensitivity and the resting membrane potential of the atrial fibroblasts declined between 8 and 30 days after infarction. The frequency of spontaneous atrial contractions was significantly reduced 8 days after myocardial infarction and recovered in parallel with the membrane potential of the fibroblasts. These findings suggest that changes in the susceptibility of atrial fibroblasts to mechanical stretch may contribute to bradyarrhythmia during postinfarct remodeling of the heart.