Stretch-dependent slow force response in isolated rabbit myocardium is Na⁺ dependent

Abstract

Objective: Stretch induces functional and trophic effects in mammalian myocardium via various signal transduction pathways. We tested stretch signal transduction on immediate and slow force response (SFR) in rabbit myocardium. Methods: Experiments were performed in isolated right ventricular muscles from adult rabbit hearts (37 °C, 1 Hz stimulation rate, bicarbonate-buffer). Muscles were rapidly stretched from 88% of optimal length (L₈₈) to near optimal length (L₉₈) for functional analysis. The resulting immediate and slow increases in twitch force (first phase and SFR, respectively) were assessed at reduced [Na⁺]_o or without and with blockade of stretch activated ion channels (SACs), angiotensin-II (AT₁) receptors, endothelin-A (ET_A) receptors, Na⁺/H⁺-exchange (NHE1), reverse mode Na⁺/Ca²⁺-exchange (NCX), or Na⁺/K⁺-ATPase. The effects of stretch on sarcoplasmic reticulum Ca²⁺-load were characterized using rapid cooling contractures (RCCs). Intracellular pH was measured in BCECF-AM loaded muscles, and action potential duration (APD) was assessed using floating electrodes. Results: On average, force increased to 216±8% of the pre-stretch value during the immediate phase, followed by a further increase to 273±10% during the SFR (n=81). RCCs significantly increased during SFR, whereas pH and APD did not change. Neither inhibition of SACs, AT₁, or ET_A receptors affected the stretch-dependent immediate phase nor SFR. In contrast, SFR was reduced by NHE inhibition and almost completely abolished by reduced [Na⁺]_o or inhibition of reverse-mode NCX, whereas increased SFR was seen after raising [Na⁺]_i by Na⁺/K⁺-ATPase inhibition. Conclusions: The data demonstrate the existence of a delayed, Na⁺- and Ca²⁺-dependent but pH and APD independent SFR to stretch in rabbit myocardium. This inotropic response appears to be independent of autocrine/paracrine AT₁ or ET_A receptor activation, but mediated through stretch-induced activation of NHE and reverse mode NCX.

Ion exchangers, Signal transduction, Stretch/m-e coupling

Time for primary review 15 days.

1 Introduction

Mammalian muscle is characterized by a biphasic force response to stretch. In isolated myocardium, stretch induces an immediate increase in twitch force (Frank-Starling mechanism), followed by a slowly developing second phase (slow force response; SFR) which was first described by Parmley and Chuck [1]. The immediate increase in force (first phase) was related to an increased sensitivity of the myofilaments for Ca²⁺, while the SFR was associated with a parallel increase in intracellular Ca²⁺ transients [2,3].

Recently, extensive work performed in isolated feline and rat heart muscle related the SFR to a stretch-dependent autocrine/paracrine release of angiotensin II (AT-II) and endothelin-1 (ET-1) with consecutive activation of the Na⁺/H⁺-exchanger (NHE) resulting in enhanced transsarcolemmal Na⁺-entry, followed by a [Na⁺]_i-dependent Ca²⁺-entry via the Na⁺/Ca²⁺-exchanger (NCX) working in its reverse mode [4–6].

However, it remains to be elucidated whether these findings may be generalized to mammalian myocardium. In addition, the explicit signal transduction pathways contributing to slow stretch-dependent inotropic effects are still unknown.

Therefore, the aim of the present study was to characterize functional effects and signal transduction pathways of stretch in isolated rabbit myocardium. The main findings of the present study were that a Na⁺- and Ca²⁺-dependent slow force response can be observed in rabbit myocardium, which is independent of changes in intracellular pH, action potential duration (APD) or activation of several receptor systems. This SFR is related to stretch-induced NHE and reverse mode NCX-activation.

2 Methods

Experiments were performed in 109 isolated right ventricular muscle strips from Chinchilla bastard rabbits. The study protocol was approved by the local ethics committee and conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996).

2.1 Muscle strip preparation

Immediately after explantation, the heart was stored in ice-cold cardioplegic Tyrode's solution containing (in mmol/l): Na⁺ 152, K⁺ 3.6, Cl⁻ 135, HCO₃⁻ 25, Mg²⁺ 0.6, H₂PO₄⁻ 1.3, SO₄²⁻ 0.6, Ca²⁺ 0.2, glucose 11.2 and 2,3-butanedione-monoxime (BDM) 30, equilibrated with carbogen (95% O₂, 5% CO₂) to a pH of 7.4. This solution has been shown to protect the myocardium from cutting injury at the time of dissection with full reversibility of the cardioplegic effects upon washout. Small trabeculae (cross-sectional area <0.5 mm²) were dissected with the help of a stereo-microscope. All preparation steps were carried out in the cardioprotective solution.

Muscles were mounted in special chambers between miniature hooks and connected to an isometric force transducer (OPT1L, Scientific Instruments, Germany) and superfused with modified Tyrode's solution of the composition given above except that BDM was omitted and insulin 10 IU/l was added. At steady-state contractile conditions, muscles were unstretched to 88% of L_max and restretched in one to 98% of L_max. A level of 98% L_max instead of 100% L_max was chosen to minimize changes in diastolic tension during the stretch protocol. To assess the role of [Na⁺]_o on functional responses, [Na⁺]_o was lowered to 76 mmol/l by replacing NaCl by LiCl. Isometric twitches were evoked through stimulation with a stimulation voltage 20% above threshold (pulse duration 5 ms) at the preload at which maximum steady-state twitch force was achieved (L_max).

2.2 Rapid cooling contractures

Rapid cooling contractures (RCCs) were elicited by a rapid decrease in the temperature of the muscle chamber from 37 to 1 °C by switching from a warm to a cold solution as previously described [7]. The cold solution was maintained at −1 °C by a cooling bath (RM20, Lauda, Lauda-Königshofen, Germany), which cools the solution and additionally surrounds the tubing that is connected to the chamber. During the cooling period the muscle was not stimulated. Rapid cooling releases all the sarcoplasmic reticulum (SR) Ca²⁺ and inhibits Ca²⁺ transport, and the resulting RCCs are an index for SR-Ca²⁺ content [8].

2.3 pH measurements

To investigate intracellular pH, muscles were loaded with BCECF-AM as described previously [9]. After loading, the muscles were attached in a specially designed set-up (Scientific Instruments) and illuminated by a 100 W mercury lamp (Ushio). The light was passed alternatively through 440- and 495-nm band-pass filters. BCECF fluorescence emitted from the muscle was directed through a 535-nm band-pass filter. The fluorescence intensities at each excitation wavelength were measured by a photomultiplier, and the fluorescence ratio F495/F440 was calculated. To minimize photobleaching, sampling intervals were selected during the protocol (20 s duration, every minute for 20 min during the first and second stretch). Twitch force and BCECF fluorescence were measured during relaxation and stretch under steady-state conditions. At the end of each experiment, fluorescence emission was calibrated by the high-K⁺ Nigericin method [10]. The calibration solution contained (mmol/l): KCl 140, MgCl₂ 1.2, Hepes 5.0, nigericin 0.01, and 2,3-BDM 30. Buffer pH was adjusted with KOH to five different values ranging from 6.8 to 7.6.

2.4 Action potential measurement

Experiments (1 Hz, 37 or 23 °C, pH 7.4) were performed in right ventricular papillary muscles or trabeculae (diameter 0.2–0.7 mm). Action potentials were recorded using 3 M KCl-filled flexible microelectrodes of approximately 10–20 MΩ resistance [11,12]. Impalement was facilitated by gently tapping the micromanipulator holding the electrode. During initial penetration the muscles were not stimulated. When resuming field stimulation stable action potential recordings could be obtained. To minimize stimulation artifacts, the pulse duration was 0.2–0.5 ms.

2.5 Drugs

To inhibit NHE HOE642 (Cariporide; Aventis Pharma, Frankfurt, Germany) was diluted from an aqueous stock solution (10 mmol/l). KB-R 7943 (Tocris, Ballwin, USA) was added from a 10 mmol/l stock (50% DMSO, 50% aqua dest.) to block reverse mode NCX. CV 11974 (Candesartan; AstraZeneca, Mölndal, Sweden) was dissolved in physiological saline and 2.5 vol.% Na₂CO₃ (10 mmol/l stock), and used at 0.1 μmol/l to block the AT₁-receptors. 0.3 μmol/l BQ123 (Calbiochem, Bad Soden, Germany) was used to block the ET_A-receptors. Gadolinium (10 μmol/l, Sigma) was used to block stretch-activated channels (SACs). Strophantidin (from a 10 mmol/l ethanol stock) was used at a final concentration of 0.1 μmol/l. All other drugs and compounds were of best analytical grade available.

2.6 Statistical analysis

Data are expressed as mean±S.E.M. Differences between basal values and values obtained after interventions were compared by two-way ANOVA analysis. Statistical significance was taken as P value of <0.05.

3 Results

3.1 Immediate and slow force response in rabbit myocardium

Rabbit myocardium is characterized by a biphasic response to stretch (see Fig. 1A): an immediate first phase, followed by a delayed slow increase in twitch force. Stretching the preparation from 88% (L₈₈) of its optimal length to L₉₈ resulted in an immediate increase in force, followed by a slowly (∼10 min) developing further rise in force (slow force response, SFR). Both phases were reproducible: After releasing the muscle back to L₈₈, a second stretch protocol revealed comparable increases in force during both phases. Average results from seven muscles are shown in Fig. 1B. The first stretch protocol resulted in an immediate increase in force to 228±17% of the pre-stretch force value during phase 1 (not shown) and a further increase to 283±19% during the SFR. This reflects a further increase in twitch force at the plateau of SFR to 124.7±2.6% of phase 1 (P<0.05, Fig. 1B). When the stretch protocol was repeated after reequilibrating the muscle at L₈₈, force increased to 231±16% and 287±17% of the unstretched value during first phase and the SFR, respectively (n.s. versus first stretch protocol). The SFR also reached the same plateau force (125±2.1% of phase 1, n.s. versus first stretch protocol, Fig. 1B). These experiments indicate the existence of a reproducible SFR in rabbit myocardium and allow further pharmacological characterisation of the mechanisms involved in the SFR. The SFR is load-dependent. We also performed experiments where we stretched muscles from 78% to 88% of L_max.This resulted in a smaller SFR (increase by 14±3 vs. 28±5% of phase 1; n=8; P<0.05 between the two stretch protocols).

Fig. 1

(A) Influence of stretch on isometric twitch force in a rabbit muscle. At steady-state conditions, the muscle was stretched from 88% of its optimal length (L₈₈) to L₉₈, resulting in an immediate and a delayed phase of force increase. After mechanical stabilisation, the muscle was unstretched to L₈₈ for 30 min, and the protocol was repeated. (B) Reproducibility of the delayed phase in force increase (2nd-1 and 2nd-2) for a stretch protocol as shown in (A). Average data from seven muscles. *P<0.05 versus force value at the end of the immediate phase (1st).

The SFR is Ca²⁺-dependent. Fig. 2 shows the effects of stretch on twitch force and rapid cooling contractures. In these experiments SFR increased to 128.7±4.6% of phase 1. This increase in force was accompanied by a parallel increase in the amplitude of the RCCs to 119.3±4.8% of the RCCs amplitude at the end of phase 1. This indicates that stretch induces enhanced SR Ca²⁺ loading during the SFR.

Fig. 2

Effect of stretch on twitch force and rapid cooling contractures (RCCs) during slow force response. Force and RCCs data were obtained in the same muscle preparations (n=7) and are related to the respective values at the end of phase 1 (1st). *P<0.05 versus 1st phase.

Fig. 3A shows representative original isometric force recordings from a rabbit papillary muscle upon stretch from L₈₈ to L₉₈. Superimposed tracings in Fig. 3B,C depict individual twitches and action potentials measured at the times indicated in Fig. 3A. Upon stretching the muscles from L₈₈ to L₉₈ force initially increased from 5.4±1.2 mN/mm² to 13.8±1.0 mN/mm². Force slowly increased further to 18.6±3.2 mN/mm² during the SFR. However, there were no significant changes in resting membrane potential, action potential duration (APD₈₀) or amplitude (Fig. 3C and Table 1).

Fig. 3

(A) Original tracing of isometric force upon stretch from 88 to 98% of L_max. (B) Individual twitches measured at the times indicated in (A). (C) Individual action potentials measured at the times indicated in (A).

Table 1

Action potential characteristics: stretched and released states of the muscle strips

37 °C (n=3)	Steady-state	Release	Stretch	Stretch
	(98% of Lmax)	(88% of Lmax)	(1st phase)	(2nd phase)
			(98% of Lmax)	(98% of Lmax)
Em resting (mV)	−76±4	−69±2	−73±1	−75±5
AP amplitude (mV)	95±5	91±8	94±1	102±3
APD80 (ms)	137±12	143±9	149±10	143±8

APD₈₀ refers to the action potential duration measured at 20% of amplitude.

Since previous experiments in cat papillary muscles at lower temperature suggested increased APD with stretch [11], we repeated similar experiments at 23 °C. Steady-state APD was increased versus 37 °C (347±24 ms vs. 143±9 ms pre-stretch), but no significant change in APD₈₀ occurred upon stretching the muscles at 23 °C (347±10 ms during phase 1 and 317±40 ms at steady state SFR). To ensure that we can readily detect changes in APD we varied frequency and rest interval. We observed the classical rabbit result of very short post-rest APD and also progressive shortening of APD at high frequency [13].

To elucidate whether stretch-activated ion channels (SAC) are involved in the SFR, muscles were preincubated with the SAC inhibitor gadolinium (10 μmol/l) for 25 min after a first stretch protocol. Gadolinium did not affect the SFR (Fig. 4A). Force increased to 125.4±2.2% before, and to 127.5±2.5% in the presence of gadolinium. In addition, SAC blockade did not affect the immediate response to stretch.

Fig. 4

(A) Influence of gadolinium (10 μmol/l; n=12, left) on SFR. Data are related to the force values at the end of the immediate phase (1st). *P<0.05 versus 1st phase. Twitch force decreased by 2±3% after administration of gadolinium (n.s.). (B) Effects of AT₁ receptor blockade with candesartan (0.1 μmol/l; n=8; left) or ET_A receptor blockade with BQ123 (0.3 μmol/l; n=8; right) on the SFR. Data are related to the force values at the end of the immediate phase (1st). *P<0.05 versus 1st phase. Twitch force increased by 4±5% after administration of Candesartan and decreased by 3±5% after administration of BQ123 (both n.s.).

An autocrine-paracrine release and action of AT II and ET-1 has been shown to underly the SFR in isolated rat and feline myocardium [4]. Therefore, we tested the role of these peptides in mediating the SFR in rabbit ventricle. Preincubation of muscle strips with the selective AT₁ receptor antagonist candesartan (0.1 μmol/l) or the selective ET_A receptor antagonist BQ123 (0.3 μmol/l) did not affect the SFR (Fig. 4B). Force increased to 129.4±4% before, and to 129.6±2.4% in the presence of Candesartan. For BQ123, force increased to 125.7±4.2% before, and to 128.4±3.7% in the presence of the ET_A-receptor blocker. To ascertain the effectiveness of the antagonists, we performed additional control experiments. Endothelin-1 (0.01 μmol/l) and angiotensin-II (10 μmol/l) exerted positive inotropic effects of 29.9±8% and 15.5±3%, respectively (both P<0.05; both n=7). BQ123 (0.3 μmol/l) and candesartan (0.1 μmol/l) completely inhibited the positive inotropic effect of 0.01 μmol/l endothelin-1 or 10 μmol/l angiotensin-II (both n=7).

3.2 Contribution of Na⁺/H⁺ exchange and Na⁺/Ca²⁺ exchange to SFR

In a further set of experiments, we assessed the contribution of NHE activation to the SFR. For this purpose, muscle strips were incubated with the selective NHE inhibitor HOE642 (10 μmol/l) for 25 min after an initial stretch protocol (Fig. 5). Before NHE inhibition, force increased to 125.3±1.9% of the phase 1 value. After incubating the preparations with HOE642 for 25 min, the SFR was reduced to 117.8±2.7 (P<0.05 versus SFR without HOE642). HOE642 did not affect the immediate increase in twitch force during phase 1 (to 209.7±16.1% without and 210.7±11.1% in the presence of HOE642, respectively).

Fig. 5

Effect of NHE1 inhibition with HOE642 (10 μmol/l; n=12) on the SFR. Data are related to the force values at the end of the immediate phase (1st). *P<0.05 versus 1st phase. ^#P<0.05 versus force increase without HOE642. Twitch force decreased by 5±3% after administration of HOE642 (n.s.).

Despite stretch-dependent NHE activation the SFR is pH-independent in physiological, bicarbonate-containing buffers. Fig. 6 summarizes experiments from six muscle preparations from six rabbit hearts showing a clear SFR within the first 5 min after stretch, without any detectable change in intracellular pH.

Fig. 6

Influence of stretch on force (bottom) and pH (top) in six muscles from six rabbit hearts. Twitch force increased from 21.4±1.8 to 25.1±2.9 mN/mm² (P<0.05) during the SFR without significant changes in pH_i. *P<0.05 versus force value at the end of the immediate phase.

Stretch-dependent NHE activation results in transsarcolemmal H⁺-elimination for Na⁺ inward transport in a 1:1 stoichiometry. In consequence both increases in pH_i and [Na⁺]_i (via reverse-mode NCX) might contribute to the SFR. Stretch-dependent NHE activation might mediate functional effects through a [Na⁺]_i-dependent activation of the reverse mode of the NCX resulting in enhanced transsarcolemmal Ca²⁺ influx. Therefore, we tested the effects of reverse-mode NCX inhibition with KB-R7943 on the SFR (Fig. 7). KB-R7943 largely reduced the SFR. Twitch force increased to 122.1±1.4% before, and to 110.9±2% in the presence of KB-R7943. As with HOE642, KB-R7943 did not affect the immediate phase in force increase upon stretch (239.19±17%, and 242.3±14.3%, respectively). KB-R7943 exerted negative inotropic effects on steady-state twitch force both at L_max and L_88% (to 66±4 and 80.2±6% of basal value, respectively).

Fig. 7

Effect of reverse-mode NCX inhibition with KB-R7943 (5 μmol/l; n=6) on the delayed phase of stretch-induced inotropy (2nd). Data are related to the force values at the end of the immediate phase (1st). *P<0.05 versus 1st phase. ^#P<0.05 versus force increase without KB-R7943. Twitch force decreased by 20±5% after administration of KB-R7943 (P<0.05).

Fig. 8 addresses the [Na⁺]_o dependence of the SFR. Lowering [Na⁺]_o to 50% of the basal value by replacing NaCl with LiCl was assumed to reduce [Na⁺]_i, whereas inhibition of the Na⁺/K⁺-ATPase with strophantidin was assumed to increase [Na⁺]_i. Initially, reduced [Na⁺]_o causes a transient increase in force for several minutes, until [Na⁺]_i declines to a new lower steady-state level [14]. The lower [Na⁺]_i is expected to limit Ca²⁺-entry via NCX, while elevating [Na⁺]_i by Na⁺/K⁺-ATPase blockade should favor Ca²⁺-entry via NCX. Low [Na⁺]_o results in an almost complete blockade of SFR (increase to 128±6.9% at 152 mmol/l [Na⁺]_o and to 105.5±3% at 76 mmol/l [Na]_o; Fig. 5 left). In contrast, 0.1 μmol/l strophantidin increased SFR by ∼20% (129.4±4.1% pre-strophantidin versus 134.7±4.6% in the presence of strophantidin; P<0.05).

Fig. 8

Effect of low [Na⁺]_o (replacing 50% of extracellular sodium by lithium; n=5; left) and Na⁺/K⁺-ATPase inhibition (strophantidin 0.1 μmol/l; n=7) on SFR. Data are related to the force values at the end of the immediate phase (1st). *P<0.05 vs. 1st phase, ^#P<0.05 vs. control.

4 Discussion

The results of the present study demonstrate that (1) stretching isolated rabbit myocardium results in an immediate, followed by a Na⁺- and Ca²⁺-dependent SFR; (2) the SFR is independent of APD and pH alterations; (3) the SFR does not rely on stretch-induced activation of AT₁ or ET_A receptors or stretch-activated ion channels; (4) both NHE and reverse mode NCX partly contribute to the SFR; (5) inhibition of the Na⁺/K⁺-ATPase stimulates the SFR. Amplitudes of SFR and contribution to total stretch-dependent increase in force were comparable to studies in other species [5,6,25].

A biphasic response to stretch was initially demonstrated by Parmley and Chuck in isolated mammalian cardiac muscle [1]. In more detailed analyses the initial response to stretch (Frank-Starling mechanism) was shown to be independent from increases in [Ca²⁺]_i and was related to enhanced responsiveness of the myofilaments for Ca²⁺ (for reviews see Fuchs and Smith [15]). In contrast, Allen and Kurihara [2] demonstrated slowly rising Ca²⁺ transients as the underlying mechanism for the SFR, but the source of elevated [Ca²⁺]_i remained obscure. Recently, the subcellular mechanism for the SFR has been further studied. Using rat and feline myocardium, Cingolani et al. reported a stretch-dependent autocrine/paracrine stimulation of angiotensin-II and endothelin-1 receptors with subsequent activation of NHE [4]. The latter resulted in elevated [Na⁺]_i leading to Ca²⁺ gain by activation of reverse mode NCX. The stretch-dependent release of angiotensin-II and endothelin-1 from isolated myocardium was in line with previous work of Sadoshima et al. in isolated rat myocytes [16]. However, it remains unknown whether the SFR is a general phenomenon in mammalian myocardium and if the same signal transduction mechanisms are always involved.

4.1 Influence of stretch on force, SR Ca²⁺ content and action potential duration in rabbit myocardium

Excitation–contraction coupling in rabbit myocardium is comparable to human myocardium [8]. However, the existence and mechanisms of SFR has never been tested previously in isolated rabbit cardiac muscle. We could clearly demonstrate a SFR in isolated rabbit myocardium. The extent of the SFR is comparable to previous work in rat and cat muscle. An increase in intracellular Ca²⁺ transients was identified as a potential mechanism for the delayed increase in stretch-dependent force [2]. However, the effect of stretch on sarcoplasmic reticulum (SR) Ca²⁺ content remains unknown. In the present study we demonstrate that RCC amplitude increases during the SFR (roughly in parallel with twitch force). Since there is no evidence for a significant increase in myofilament Ca²⁺ sensitivity during the SFR (and no pH change during the SFR), this is consistent with a gradual gain in SR Ca²⁺ as a central mechanism underlying the SFR.

Both, AP prolongation [11] and AP shortening [17–19] have been described following stretch in mammalian myocardium. Since Ca²⁺ entry through voltage-dependent Ca²⁺ channels occurs during depolarisation, a stretch-dependent prolongation of APD might underly the SFR and the increase in SR-Ca²⁺ observed in the present study. In addition, prolonged depolarisation might also favor reverse-mode NCX Ca²⁺ entry [27]. However, we did not observe any effect of stretch on APD or resting cell membrane potential in isolated rabbit trabeculae. This is in line with previous work in isolated sheep Purkinje fibers [20] and Langendorff perfused canine hearts [21].

4.2 Signal transduction of the SFR

Sarcolemmal stretch-activated ion channels are potential candidates to mediate the increase in intracellular Ca²⁺ and SFR (for review, see Hu and Sachs [22]). We used gadolinium to block cation SACs. Gadolinium suppresses stretch-induced transient depolarizations and premature beats [23] and prevents stretch-mediated contractile dysfunction in guinea pig papillary muscle [24]. However, pretreatment of muscles with gadolinium did not affect the immediate force increase or SFR upon stretch in the present study. This indicates that stretch-activated ion channels are not involved in stretch-induced inotropy in rabbit myocardium.

In 1993, Sadoshima et al. reported a stretch-dependent release of angiotensin-II and endothelin-1 from neonatal rat myocytes [16]. This work was extended to adult rat and feline myocardium by Cingolani and co-workers [4–6]. In their initial report, these authors demonstrated in cat papillary muscle a stretch-dependent intracellular alkalinisation which could be blocked by either the AT₁ receptor antagonist losartan or the ET_A receptor antagonist BQ123. These authors concluded that stretching feline (or rat) cardiac muscle releases AT II which induces secretion of ET-1 in a autocrine/paracrine manner. However, recently Calaghan and White were not able to block the SFR with losartan or BQ123 in ferret papillary muscle [25]. In the present work, neither AT₁ receptor blockade with candesartan nor ET_A receptor blockade with BQ123 prevented the immediate or slow force response. It has been shown that, in rabbit myocardium, ET-1 inotropism is partly mediated via the ET_A2 subtype, which is less sensitive to BQ123 [26]. However, 0.3 μM BQ123 did entirely block the positive inotropic effect of endothelin-1 in our control experiments. Therefore, stretch-dependent acute release of functional amounts of angiotensin-II and endothelin-1 are not of major relevance for the SFR in rabbit myocardium under our experimental conditions. Therefore, we suggest that species-dependent differences exist with respect to the contribution of neuroendocrine peptides to the slow force response: while autocrine/paracrine angiotensin and endothelin release mediates the SFR in rat and cat cardiac muscle, this is not of major relevance in rabbit and ferret. The SFR is also present in failing human myocardium (see the review by Cingolani et al. in the present spotlight issue). Interestingly, angiotensin II does not exert inotropic effects in human ventricular muscle (own observation), and accordingly, we did not detect an effect of angiotensin receptor blockade on the SFR in isolated human ventricular trabeculae in preliminary experiments.

Stretch-dependent inotropic effects due to NHE activation were related to intracellular alkalinisation with enhanced myofilament responsiveness for Ca²⁺ or increases in [Na⁺]_i[4] which thermodynamically favors the NCX to operate in its reverse mode [27]. The importance of intracellular alkalinisation for inotropic responses after stretch-induced NHE activation has been recently challenged and depends on the use of unphysiological, bicarbonate-free buffers [28]. In fact, Alvarez et al. [5], using CO₂/HCO₃⁻ buffered medium, demonstrated a stretch-induced NHE-activation with consecutive increases in [Na⁺]_i, [Ca²⁺]_i and twitch force without changes in pH_i in rat trabeculae at room temperature. Presumably in HCO₃⁻ solutions the ability of Na-HCO₃⁻ cotransport and Cl⁻–HCO₃⁻ exchange counteract the elimination of H⁺ and prevent significant alkalinisation, despite NHE activation. Our experiments in rabbit myocardium confirm these observations at physiological temperatures and also indicate that changes in pH are not a prerequisite for the SFR. The loss of cellular protons due to NHE activation was most likely couterbalanced by bicarbonate-dependent anion exchangers resulting in unchanged pH_i. One possible explanation is that NHE (or an intrinsic regulator of NHE) acts as a sarcolemmal mechanosensor which directly or indirectly converts mechanical stress into functional responses.

Perez et al. [6] first described the dependence of the SFR on the activation of the NCX in its reverse mode (based on studies with KB-R7943). The activation of the NCX reverse mode would lead to cellular Ca²⁺ gain (increasing force) coupled to outward transport of Na⁺ (which depends on [Na⁺]_i). Therefore, reverse-mode NCX activity may be depressed by depletion of [Na⁺]_i (as a consequence of reduced [Na⁺]_o), and enhanced by elevation of [Na⁺]_i (strophantidin). This is consistent with our results where the SFR was reduced by low [Na⁺]_i and enhanced by strophantidin (Fig. 8). The results with the pharmacological reverse-mode NCX blocker KB-R7943 [29,30] (Fig. 7) are also consistent with this interpretation. However, KB-R7943 is not an ideal agent, since it can also affect other transport systems such as K⁺, Na⁺ and Ca²⁺ channels and affects Ca²⁺ transients, even in NCX-knockout heart tubes [31]. The apparent selectivity for outward versus inward NCX current is not well understood [32,33]. Nevertheless, the balance of data here concerning SFR favors a mechanism where there is cellular and SR Ca²⁺ gain that depends on a shift in Ca²⁺ fluxes via NCX, which in turn depends on elevated [Na⁺]_i via enhanced NHE activity.

Therefore, we conclude that NHE activation mediates a part of the SFR as a consequence of NHE-dependent increase in [Na⁺]_i, followed by the observed increase in [Ca²⁺]_i through reverse mode NCX activation. Both direct activation by mechanical signals or indirect activation through distinct mechanosensors, such as focal adhesion kinase-mediated phosphorylation might contribute to NHE activation [34,35]. We cannot exclude the possibility of stretch-induced leak of protons or Na⁺ into cells, which could still result in activation of the same NHE and NCX cascade. Leaks small enough to not alter pH_i, membrane potential or action potential characteristics could still cause the SFR associated changes in [Na⁺]_i and Ca²⁺ flux.

The present data demonstrate the existence of a second mechanism of force recruitment besides the Frank-Starling-mechanism. This SFR is increasing towards the upper end of the length tension curve (own unpublished data). In addition, it develops gradually over a prolonged period of time. Under functional aspects, the SFR might serve as an additional ‘reserve’ mechanism to increase contractile strength of the heart during states of hemodynamic overload. However, in contrast to the FSM [36] increased intracellular Ca²⁺ cycling is involved in the SFR which is energetically unfavourable. In addition, trophic consequences of stretch-dependent NHE and reverse-mode NCX activation need to be discussed. Stretch is a major stimulus for growth, and NHE inhibition has been shown to inhibit myocardial hypertrophy and fibrosis [37]. Reverse-mode NCX activation with a more tonic increase in [Ca²⁺]_i may—via Ca²⁺-dependent hypertrophic signaling cascades, such as calcineurin/NFAT—translate stretch to myocardial hypertrophy.

In conclusion, we demonstrated the existence of a delayed inotropic response to stretch in rabbit myocardium (SFR). This SFR depends on [Na⁺] and is associated with increased intracellular Ca²⁺. Changes in APD, SACs or intracellular pH do not contribute to the SFR, and neither AT₁ nor ET_A receptor activation appears to be required. Rather the SFR appears to be related to stretch-dependent activation of NHE (direct or indirect) and reverse mode NCX (at least in part secondary to increased [Na⁺]_i).

Acknowledgements

This work was supported by a grant from the Deutsche Forschungsgemeinschaft to Dr. Pieske (PI-414/1). Dr. Maier is in the Emmy-Noether-Program of the DFG (MA 1982/1-1).