Regulation of the cardiac sodium pump.

In cardiac muscle, the sarcolemmal sodium/potassium ATPase is the principal quantitative means of active transport at the myocyte cell surface, and its activity is essential for maintaining the trans-sarcolemmal sodium gradient that drives ion exchange and transport processes that are critical for cardiac function. The 72-residue phosphoprotein phospholemman regulates the sodium pump in the heart: unphosphorylated phospholemman inhibits the pump, and phospholemman phosphorylation increases pump activity. Phospholemman is subject to a remarkable plethora of post-translational modifications for such a small protein: the combination of three phosphorylation sites, two palmitoylation sites, and one glutathionylation site means that phospholemman integrates multiple signaling events to control the cardiac sodium pump. Since misregulation of cytosolic sodium contributes to contractile and metabolic dysfunction during cardiac failure, a complete understanding of the mechanisms that control the cardiac sodium pump is vital. This review explores our current understanding of these mechanisms.

Na(V)1.5 enhances breast cancer cell invasiveness by increasing NHE1-dependent H(+) efflux in caveolae.

Na(V)1.5 sodium channels enhance the invasiveness of breast cancer cells through the acidic-dependent activation of cysteine cathepsins. Here, we showed that the Na(+)/H(+) exchanger type 1 (NHE1) was an important regulator of H(+) efflux in breast cancer cells MDA-MB-231 and that its activity was increased by Na(V)1.5. Na(V)1.5 and NHE1 were colocalized in membrane rafts containing caveolin-1. The inhibition of Na(V)1.5 or NHE1 induced a similar reduction in cell invasiveness and extracellular matrix degradation; no additive effect was observed when they were simultaneously inhibited. Our study suggests that Na(V)1.5 and NHE1 are functionally coupled and enhance the invasiveness of cancer cells by increasing H(+) efflux.

Transmural variations in gene expression of stretch-modulated proteins in the rat left ventricle.

The properties of left ventricular cardiac myocytes vary transmurally. This may be related to the gradients of stress and strain experienced in vivo across the ventricular wall. We tested the hypothesis that within the rat left ventricle there are transmural differences in the expression of genes for proteins that are involved in mechanosensitive pathways and in associated physiological responses. Real time reverse transcription polymerase chain reaction was used to measure messenger RNA (mRNA) levels of selected targets in sub-epicardial (EPI) and sub-endocardial (ENDO) myocardium. Carbon fibres were attached to single myocytes to stretch them and to record contractility. We observed that the slow positive inotropic response to stretch was not different between EPI and ENDO myocytes and consistent with this, that the mRNA expression of two proteins implicated in the slow response, non-specific cationic mechanosensitive channels (TRPC-1) and Na/H exchanger, were not different. However, mRNA levels of other targets, e.g. the mechanosensitive K(+) channel TREK-1, Brain Natriuretic Peptide and Endothelin-1 receptor B, were significantly greater in ENDO than EPI. No targets had significantly greater mRNA levels in EPI than ENDO. On the basis of these findings, we suggest that the response of the ventricle to stretch will depend upon both the regional differences in stimuli and the relative expression of the mechanosensitive targets and that generally, stretch sensitivity is predicted to be greater in ENDO.

Cytoskeletal modulation of electrical and mechanical activity in cardiac myocytes.

The cardiac myocyte has an intracellular scaffold, the cytoskeleton, which has been implicated in several cardiac pathologies including hypertrophy and failure. In this review we describe the role that the cytoskeleton plays in modulating both the electrical activity (through ion channels and exchangers) and mechanical (or contractile) activity of the adult heart. We focus on the 3 components of the cytoskeleton, actin microfilaments, microtubules, and desmin filaments. The limited visual data available suggest that the subsarcolemmal actin cytoskeleton is sparse in the adult myocyte. Selective disruption of cytoskeletal actin by pharmacological tools has yet to be verified in the adult cell, yet evidence exists for modulation of several ionic currents, including I(CaL), I(Na), I(KATP), I(SAC) by actin microfilaments. Microtubules exist as a dense network throughout the adult cardiac cell, and their structure, architecture, kinetics and pharmacological manipulation are well described. Both polymerised and free tubulin are functionally significant. Microtubule proliferation reduces contraction by impeding sarcomeric motion; modulation of sarcoplasmic reticulum Ca(2+) release may also be involved in this effect. The lack of effect of microtubule disruption on cardiac contractility in adult myocytes, and the concentration-dependent modulation of the rate of contraction by the disruptor nocodazole in neonatal myocytes, support the existence of functionally distinct microtubule populations. We address the controversy regarding the stimulation of the beta-adrenergic signalling pathway by free tubulin. Work with mice lacking desmin has demonstrated the importance of intermediate filaments to normal cardiac function, but the precise role that desmin plays in the electrical and mechanical activity of cardiac muscle has yet to be determined.

Do stretch-induced changes in intracellular calcium modify the electrical activity of cardiac muscle?

Stretch of the myocardium influences the shape and amplitude of the intracellular Ca(2+)([Ca(2+)](i)) transient. Under isometric conditions stretch immediately increases myofilament Ca(2+) sensitivity, increasing force production and abbreviating the time course of the [Ca(2+)](i) transient (the rapid response). Conversely, muscle shortening can prolong the Ca(2+) transient by decreasing myofilament Ca(2+) sensitivity. During the cardiac cycle, increased ventricular dilation may increase myofilament Ca(2+) sensitivity during diastolic filling and the isovolumic phase of systole, but enhance the decrease in myofilament Ca(2+) sensitivity during the systolic shortening of the ejection phase. If stretch is maintained there is a gradual increase in the amplitude of the Ca(2+) transient and force production, which takes several minutes to develop fully (the slow response). The rapid and slow responses have been reported in whole hearts and single myocytes. Here we review stretch-induced changes in [Ca(2+)](i) and the underlying mechanisms. Myocardial stretch also modifies electrical activity and the opening of stretch-activated channels (SACs) is often used to explain this effect. However, the myocardium has many ionic currents that are regulated by [Ca(2+)](i) and in this review we discuss how stretch-induced changes in [Ca(2+)](i) can influence electrical activity via the modulation of these Ca(2+)-dependent currents. Our recent work in single ventricular myocytes has shown that axial stretch prolongs the action potential. This effect is sensitive to either SAC blockade by streptomycin or the buffering of [Ca(2+)](i) with BAPTA, suggesting that both SACs and [Ca(2+)](i) are important for the full effects of axial stretch on electrical activity to develop.

Cardiac microtubules are more resistant to chemical depolymerisation in streptozotocin-induced diabetes in the rat.

Evidence exists for a specific diabetic cardiomyopathy independent of concurrent vascular disease. Our aim was to test the hypothesis that a change in the microtubular cytoskeleton may contribute to cardiac dysfunction in type-1 diabetes. Resting sarcomere length and characteristics of unloaded shortening were measured in ventricular myocytes from rats 2 months after injection of streptozotocin (STZ). Microtubular density and organisation were assessed using immunofluorescence confocal microscopy and the effects of microtubule disruption by colchicine on shortening and microtubules were examined. Diabetic myocytes showed a significant reduction in resting sarcomere length and a 30% increase in time to peak shortening. The microtubule disruptor colchicine (10 micromol/l) had no effect on the amplitude or kinetics of shortening in myocytes from control or diabetic rats. Cardiac microtubular density and organisation were similar in control and diabetic animals, yet although colchicine significantly reduced microtubule density in control myocytes, microtubules in diabetic myocytes were resistant to its effects. These observations of an increase in microtubular stability in STZ-diabetes of 2 months duration imply a disruption to the normal balance between populations of dynamic and drug-stable microtubules. Such disruption has been observed in other pathological conditions and may contribute to diabetic cardiomyopathy.

Modulation of Ca(2+) signaling by microtubule disruption in rat ventricular myocytes and its dependence on the ruptured patch-clamp configuration.

In the absence of hypertrophic proliferation of microtubules, microtubule disruption by colchicine does not modulate contraction of adult cardiac myocytes. However, Gomez et al (Circ Res. 2000;86:30-36) recently reported that disruption of microtubules by colchicine in ruptured patch-clamped myocytes increased I(Ca,L) density and [Ca(2+)](i) transient amplitude and depressed the response of these parameters to the beta-adrenoceptor agonist isoproterenol. These effects were ascribed to stimulation of adenylyl cyclase by increased intracellular free tubulin. In the present study, we show that in intact rat ventricular myocytes, 2 to 4 hours of exposure to 10 micromol/L colchicine had no effect on shortening or [Ca(2+)](i) transient amplitude or on the amplitude of I(Ca,L) in perforated patch-clamped cells, under basal conditions and after stimulation with 1 micromol/L isoproterenol. However, in ruptured patch-clamped myocytes, basal I(Ca,L) was 2-fold higher after treatment with colchicine compared with vehicle and, in contrast to vehicle-treated cells, I(Ca,L) did not increase in response to isoproterenol. Cell width decreased during ruptured patch-clamp experiments in colchicine-treated but not vehicle-treated myocytes. We conclude that in cells with intact sarcolemma, colchicine does not modulate Ca(2+) signaling or the response to beta stimulation. However, the combination of microtubule disruption by colchicine and the ruptured patch configuration activates I(Ca,L) and attenuates the response to beta stimulation. We propose that these effects may be due to loss of free tubulin by intracellular dialysis or to increased sensitivity to mechanical stimulation as a result of microtubule disruption. These findings have important implications for cardiomyopathies associated with decreased free tubulin or a diminished microtubular network. The full text of this article is available at http://www.circresaha.org.

Contribution of angiotensin II, endothelin 1 and the endothelium to the slow inotropic response to stretch in ferret papillary muscle.

We investigated the contribution of angiotensin II and endothelin I to the slow positive inotropic response observed following stretch of isolated ferret papillary muscle from 88% to 98% of the length at which maximum force is generated. Angiotensin antagonists losartan and saralasin did not affect the magnitude of the slow response in ferret papillary muscle. The ETA-selective antagonist BQ123 slightly reduced the magnitude of the slow response (P > 0.05). In the presence of PD145065 (an ETA and ETB antagonist), the magnitude of the slow response was reduced significantly by 50%. Removal of the endothelium with 1% Triton X-100 reversed the slow response to stretch. We conclude that, in the ferret, endothelin I acting through ETA and ETB receptors, contributes to the slow response although it is not the sole mediator. Angiotensin II is not a prerequisite for the slow response to stretch. We have shown for the first time that the endocardial endothelium plays a pivotal role in this phenomenon in cardiac papillary muscle.

Cytochalasin D reduces Ca2+ sensitivity and maximum tension via interactions with myofilaments in skinned rat cardiac myocytes.

The F-actin disrupter cytochalasin D depresses cardiac contractility, an effect previously ascribed to the interaction of cytochalasin D with cytoskeletal actin. We have investigated the possibility that this negative inotropic effect is due to the interaction of cytochalasin D with sarcomeric actin of the thin filament. Confocal images of Triton X-100-skinned myocytes incubated with a fluorescent conjugate of cytochalasin D revealed a longitudinally striated pattern of binding, consistent with a myofibrillar rather than cytoskeletal structure.Tension-pCa relationships were determined at sarcomere lengths (SLs) of 2.0 and 2.3 [mu]m following 2 min incubation with 1 [mu]M cytochalasin D. Cytochalasin D significantly reduced the pCa for half-maximal activation (pCa50) at both SLs. The shift in pCa50 was significantly greater at a SL of 2.3 [mu]m compared with that at a SL of 2.0 [mu]m. Cytochalasin D had no effect on the Hill co-efficient at either SL. Cytochalasin D significantly reduced the maximum tension at both SLs. We suggest that the length-dependent decrease in myofilament Ca2+ sensitivity in response to cytochalasin D is due to a decrease in the affinity of troponin C for Ca2+. Cytochalasin D has been used for many years as the agent of choice for disruption of cytoskeletal actin. However, we have demonstrated for the first time an interaction of cytochalasin D with sarcomeric actin of the thin filament, which can account for the effects of cytochalasin D on cardiac contractility.

A role for C-protein in the regulation of contraction and intracellular Ca2+ in intact rat ventricular myocytes.

1. C-protein is a major component of muscle thick filaments whose function is unknown. We have examined for the first time the role of the regulatory binding domain of C-protein in modulating contraction and intracellular Ca2+ concentration ([Ca2+]i) in intact cardiac myocytes. 2. Rat ventricular myocytes were reversibly permeabilised with the pore-forming toxin streptolysin O. Myosin S2 (which binds to the regulatory domain of C-protein) was introduced into cells during permeabilisation to compete with the endogenous C-protein-thick filament interaction. 3. Introduction of S2 into myocytes increased contractility by approximately 30%, significantly lengthened the time to peak of the contraction and the time to half-relaxation, but had no effect on [Ca2+]i transient amplitude. 4. Our data are consistent with increased myofilament Ca2+ sensitivity when there is reduced binding of C-protein to myosin near the head-tail junction. 5. We propose that the effects of introducing S2 into intact cardiac cells can be equated with the consequences of selectively phosphorylating C-protein in vivo, and that the regulation of contraction by C-protein is mediated by the effects of crossbridge cycling on the Ca2+ affinity of troponin C.

Biphasic effects of hyposmotic challenge on excitation-contraction coupling in rat ventricular myocytes.

The effects of short (1 min) and long (7-10 min) exposure to hyposmotic solution on excitation-contraction coupling in rat ventricular myocytes were studied. After short exposure, the action potential duration at 90% repolarization (APD(90)), the intracellular Ca(2+) concentration ([Ca(2+)](i)) transient amplitude, and contraction increased, whereas the L-type Ca(2+) current (I(Ca, L)) amplitude decreased. Fractional sarcoplasmic reticulum (SR) Ca(2+) release increased but SR Ca(2+) load did not. After a long exposure, I(Ca,L), APD(90), [Ca(2+)](i) transient amplitude, and contraction decreased. The abbreviation of APD(90) was partially reversed by 50 microM DIDS, which is consistent with the participation of Cl(-) current activated by swelling. After 10-min exposure to hyposmotic solution in cells labeled with di-8-aminonaphthylethenylpyridinium, t-tubule patterning remained intact, suggesting the loss of de-t-tubulation was not responsible for the fall in I(Ca,L). After long exposure, Ca(2+) load of the SR was not increased, and swelling had no effect on the site-specific phosphorylation of phospholamban, but fractional SR Ca(2+) release was depressed. The initial positive inotropic response to hyposmotic challenge may be accounted for by enhanced coupling between Ca(2+) entry and release. The negative inotropic effect of prolonged exposure can be accounted for by shortening of the action potential duration and a fall in the I(Ca,L) amplitude.

Cyclic AMP but not phosphorylation of phospholamban contributes to the slow inotropic response to stretch in ferret papillary muscle.

cAMP has been suggested to mediate the increased intracellular Ca2+ transient and contraction seen during the slow response to stretch in cardiac muscle. We measured cAMP in ferret papillary muscles stretched from 80-85% to 98% of their length at which maximum active tension is produced (Lmax) for 15 min. cAMP was significantly (P<0. 05) increased by 53% in muscles at the longer length which showed the slow response compared with controls. By contrast, in a population of muscles that were stretched but did not show the slow response, cAMP was not significantly different from that in muscles at the short length. Although cAMP can increase sarcoplasmic reticulum (SR) Ca2+ uptake by phosphorylation of phospholamban, we found no significant effect of stretch on phosphorylation of phospholamban at either Ser16 or Thr17. Further support for the hypothesis that cAMP is a mediator of the slow response was obtained by exposure of some muscles to the cell-permeable cAMP antagonist 8-bromo, adenosine 3',5'-cyclic monophosphorothioate, Rp isomer (Rp-8-Br-cAMPS, (2.5-10 microM). The slow response was reduced by 30% (P<0.05) in the presence of this antagonist. Our results not only provide evidence for the mediation of the slow response to stretch by cAMP, they also suggest that cAMP may rise in an intracellular compartment inaccessible to the SR.

Effect of the microtubule polymerizing agent taxol on contraction, Ca2+ transient and L-type Ca2+ current in rat ventricular myocytes.

1. Microtubules form part of the cytoskeleton. Their role in adult ventricular myocytes is not well understood although microtubule proliferation has previously been linked with reduced contractile function. 2. We investigated the effect of the anti-tumour drug taxol, a known microtubule polymerizing agent, on Ca2+ handling in adult rat ventricular myocytes. 3. Treatment of cells with taxol caused proliferation of microtubules. 4. In taxol-treated cells there was a reduction in the amplitude of contraction, no significant effect on the amplitude of L-type Ca2+ current, but a significant reduction in the amplitude of the Ca2+ transient. 5. Caffeine was used to release Ca2+ from the sarcoplasmic reticulum (SR). There was a significant reduction in the ratio of electrically stimulated : caffeine-induced Ca2+ transients in taxol-treated cells. This observation is consistent with the hypothesis that taxol reduces fractional SR Ca2+ release. 6. We suggest that the negative inotropic effect of taxol may, at least in part, be the result of reduced release of Ca2+ from the SR. Microtubules may be important regulators of Ca2+ handling in the heart.

The role of calcium in the response of cardiac muscle to stretch.

This review focuses on the complex interactions between two major regulators of cardiac function; Ca2+ and stretch. Initial consideration is given to the effect of stretch on myocardial contractility and details the rapid and slow increases in contractility. These are shown to be related to two diverse changes in Ca2+ handling (enhanced myofilament Ca2+ sensitivity and increased intracellular Ca2+ transient, respectively). Interaction between stretch and Ca2+ is also demonstrated with respect to the rhythm of cardiac contraction. Stretch has been shown to alter action potential configuration, generate stretch-activated arrhythmias, and increase the rate of beating of the sino-atrial node. A variety of Ca(2+)-dependent mechanisms including attenuation of Ca2+ extrusion via Na+/Ca2+ exchange, Ca2+ entry through stretch-activated channels (SACs) and mobilisation of intracellular Ca2+ stores have been proposed to account for the effect of stretch on rhythm. Finally, the interaction between stretch and Ca2+ in the secretion of natriuretic peptides and onset of hypertrophy is discussed. Evidence is presented that Ca2+ (entering through L-type Ca2+ channels or SACs, or released from sarcoplasmic reticular stores) influences secretion of both atrial and B-type natriuretic peptide; there is data to support both positive and negative modulation by Ca2+. Ca2+ also appears to be important in the pathway that leads to expression of precursors of hypertrophic protein synthesis. In conclusion, two of the major regulators of cardiac muscle function, Ca2+ and stretch, interact to produce effects on the heart; in general these effects appear to be additive.

Co-ordinated changes in cAMP, phosphorylated phospholamban, Ca2+ and contraction following beta-adrenergic stimulation of rat heart.

Concentration-dependent changes in cyclic AMP (cAMP), site-specific phosphorylation of phospholamban, the intracellular calcium ([Ca2+]i) transient and contraction were measured in isolated rat ventricular myocytes exposed to the beta-adrenoceptor agonist isoprenaline. Cyclic AMP was measured by [125I]-cAMP scintillation proximity assay, phosphorylation of phospholamban at Ser16 and Thr17 was assessed using a pair of site-specific polyclonal antibodies, and [Ca2+]i was monitored with the fluorescent dye fura 2. Cyclic AMP rose to twice basal levels in the presence of 10(-6) M isoprenaline. The maximum increase in phosphorylation at Ser16 and Thr17 of phospholamban was seen at 10(-7) M isoprenaline. At this stage Ser16 phosphorylation was six times higher, and Thr17 phosphorylation was three times higher than that recorded in the absence of isoprenaline. Phosphorylation at Ser16 correlated more closely with changes in the [Ca2+]i transient and contraction than did phosphorylation at Thr17. This is the first study of its kind to measure simultaneous changes in cAMP, the phosphorylation of phospholamban, the [Ca2+]i transient and contraction over a range of concentrations of beta-agonist. The results suggest that phosphorylation of phospholamban at Thr17 is of lesser physiological relevance to the effects of beta-adrenergic stimulation on the heart than phosphorylation at Ser16.

Preservation of the in vivo phosphorylation status of phospholamban in the heart: evidence for a site-specific difference in the dephosphorylation of phospholamban.

The phosphorylation status of the cardiac sarcoplasmic reticular (SR) protein phospholamban determines the activity of the SR Ca(2+)-ATPase. In order to predict SR Ca2+ transport in vivo, it is vital that techniques used to measure the phosphorylation status of phospholamban adequately clamp the endogenous kinases and phosphatases which modify phosphorylation during sample preparation. A recent study (Boateng, S., Seymour, A-M., Dunn, M., Yacoub, M., and Boheler, K. (1997) Biochem. Biophys. Res. Comm. 239, 701-705) has suggested that phosphatase inhibitors must be present in quenching media to prevent almost total dephosphorylation of phospholamban. We addressed this issue by assessing the effect of both kinase and phosphatase inhibition on site-specific phosphorylation of phospholamban in ferret ventricular muscle and isolated rat ventricular myocytes quenched with Laemmli sample buffer. Under these clearly defined quenching conditions in isolated myocytes, we demonstrated that the phosphorylation status of phospholamban was low under basal conditions, and high following exposure to the beta-agonist isoprenaline. The only significant effect of inhibitor inclusion in the quench solution was in isolated myocyte preparations where phosphatase inhibition increased phosphorylation at Ser16 by about a third. The differential effect of phosphatase inclusion on phosphorylation at Ser16 and Thr17 may indicate that different enzymes are involved in dephosphorylation of the two sites.