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(Circulation Research. 2003;92:169.)
© 2003 American Heart Association, Inc.

Integrative Physiology

Insulin-Like Growth Factor-1 Exerts Ca²⁺-Dependent Positive Inotropic Effects in Failing Human Myocardium

Dirk von Lewinski, Kerstin Voß, Swen Hülsmann, Harald Kögler, Burkert Pieske

From the Abteilung Kardiologie und Pneumologie (D.v.L., K.V., H.K., B.P.) and Abteilung Neuro- und Sinnesphysiologie (S.H.), Georg-August-Universität Göttingen, Germany.

Correspondence to Prof Dr Burkert Pieske, Abteilung Kardiologie und Pneumologie, Georg-August-Universität Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany. E-mail pieske{at}med.uni-goettingen.de

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Myocardial generation of insulin-like growth factor-1 (IGF-1) is altered in hypertrophy and heart failure, but there are no reports on acute functional effects of IGF-1 in human cardiac muscle. We examined inotropic responses and signal transduction mechanisms of IGF-1 in human myocardium. Experiments were performed in isolated trabeculae or cardiomyocytes from 46 end-stage failing hearts. The effect of IGF-1 (0.001 to 0.2 µmol/L) on isometric twitch force (37°C, 1 Hz), intracellular Ca²⁺ transients (aequorin method), sarcoplasmic reticulum (SR) Ca²⁺ content (rapid cooling contractures), L-type Ca²⁺ current (whole-cell voltage clamp), and cAMP concentrations was assessed. In addition, the effects of blocking IGF-1 receptors, phosphoinositide 3-kinase (PI3-kinase), protein kinase C (PKC), or transsarcolemmal Ca²⁺ entry were tested. IGF-1 exerted concentration-dependent positive inotropic effects (twitch force increased to maximally 133±4% of baseline values at 0.1 µmol/L; P<0.05). The IGF-1 receptor antibody IR3 or the PI3-kinase inhibitor wortmannin prevented the functional effects. The inotropic response was paralleled by increases in Ca²⁺ transients and SR Ca²⁺ content. IGF-1 (0.1 µmol/L) increased L-type Ca²⁺ current amplitude by 24±7% (P<0.05). Blockade of SR function did not affect the inotropic response to IGF-1. In contrast, L-type Ca²⁺ channel blockade with diltiazem partially prevented (50%) the inotropic response to IGF-1. Inhibition of PKC (GF109203X), Na⁺-H⁺ exchange (HOE642), or reverse-mode Na⁺-Ca²⁺ exchange (KB-R7943) reduced the response to IGF-1 by 60% to 70%. IGF-1 exerts Ca²⁺-dependent positive inotropic effects through activation of IGF-1 receptors and a PI3-kinase-dependent pathway in failing human myocardium. The increased [Ca²⁺]_i with IGF-1 originates from both enhanced L-type Ca²⁺ currents and enhanced Na⁺-H⁺ exchange-dependent reverse-mode Na⁺-Ca²⁺ exchange. These nongenomic functional effects of IGF-1 may be of clinical relevance.

Key Words: insulin-like growth factor-1 • functional effects • Ca²⁺ handling • heart failure • human myocardium

	Introduction

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Insulin-like growth factor-1 (IGF-1), a circulating and locally produced peptide hormone structurally and functionally related to insulin, is synthesized under the control of growth hormone (GH) by various cell types, including cardiac myocytes.¹ IGF-1 stimulates protein synthesis and growth and exerts metabolic as well as antiapoptotic effects in many organs including cardiac muscle.^2–4 Recently, increased local cardiac IGF-1 release and increased cardiac IGF-1 mRNA production and IGF-1 receptor expression were reported in heart failure animal models and in human cardiac diseases.^5–7

Normal activation of the GH-IGF axis is essential for myocardial performance,⁸ and GH/IGF-1 deficiency is associated with impaired cardiac function.⁹ Chronic GH substitution may induce beneficial long-term effects on myocardial function in GH deficiency⁸ and chronic heart failure.^10,11 Interestingly, acute application of IGF-1 also enhanced circulatory function with increased stroke volume and cardiac output in healthy volunteers and patients with chronic heart failure.^12,13 However, IGF-1 exerts endothelium-dependent vasodilatory actions,^14,15 and it remains unclear whether improved hemodynamics result from direct inotropic or peripheral vasodilatory effects of IGF-1.

A limited number of investigations have directly tested the acute functional effects of IGF-1 in mammalian myocardium. In these studies, IGF-1 increased contractility in isolated rat,¹⁶ ferret,¹⁷ or dog¹⁸ myocardium, but the subcellular mechanism of action of IGF-1 remained controversial.^17,18 No data are available on functional effects of IGF-1 in human cardiac muscle.

Therefore, we directly assessed functional effects and mechanisms of action of IGF-1 in isolated failing human myocardium. Our main finding was that IGF-1 exerts receptor-mediated, Ca²⁺-dependent positive inotropic effects that are related to a phosphoinositide 3-kinase (PI3-kinase)-dependent activation of L-type Ca²⁺ currents and Na⁺-H⁺ exchange.

	Materials and Methods

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Human Myocardium
Experiments were performed in muscle strips obtained from 46 end-stage failing hearts (from 14 female and 32 male patients). The mean ejection fraction before transplantation was 23.4±0.8%. The study protocol was approved by the local ethics committee. For details, please refer to the expanded Materials and Methods section available in the online data supplement at http://www.circresaha.org.

Muscle Strip Preparation
Small endocardial trabeculae were dissected from the left or right ventricle as previously described,¹⁹ connected to an isometric force transducer, and superfused with bicarbonate-containing Tyrode’s solution. Muscles were field stimulated at 1 Hz (37°C), and isometric contractions were recorded at optimal preload (L_max). The functional effects of IGF-1 were assessed by cumulative concentration-response curves (0.001 to 0.2 µmol/L) or by a single, maximally effective concentration of IGF-1 (0.1 µmol/L).

Aequorin Measurements
At steady-state contractile function, the Ca²⁺-regulated bioluminescent photoprotein aequorin was macroinjected into the quiescent muscle as described previously.¹⁹ Aequorin light emission was detected using a photomultiplier, which was vertically mounted with its cathode just above the glass cuvette containing the muscle.

Rapid Cooling Contractures (RCCs)
RCCs were elicited by a rapid decrease in the temperature of the muscle chamber from 37°C to 1°C by switching from a warm to a cold solution with solenoid pinch valves at the bath inlet as previously described.²⁰ The resulting cooling contracture is an index for sarcoplasmic reticulum (SR) Ca²⁺ content.

Patch-Clamp Experiments
Human ventricular myocytes were isolated with collagenase.²¹ Ca²⁺ currents were recorded using the whole-cell patch-clamp technique.²² Myocytes were placed in a 0.5-mL chamber and superfused with Tyrode’s solution (30°C). Currents were recorded using a Multiclamp 700 patch-clamp amplifier (Axon Instruments). The peak inward current during a depolarization step from -40 to +10 mV was taken as I_Ca,L. At steady-state basal current recordings, IGF-1 (0.1 µmol/L) was added to the superfusate, and the effects of IGF-1 on Ca²⁺ currents were analyzed.

cAMP Determinations
Intracellular cAMP levels in homogenates of IGF-1- or isoproterenol (ISO)-prestimulated muscle strips were measured by a commercially available [³H]cAMP assay system (Amersham Pharmacia) according to the protocol provided by the manufacturer.

Drugs
Recombinant human IGF-1 (Sigma) or IGF-1 receptor antibody (IR-3 clone; Oncogene Research Products) was dissolved in Tyrode’s solution and added to the organ bath. Diltiazem (Sigma), HOE642 (Cariporide; a gift of Aventis Pharma), KB-R7943 (Tocris), or wortmannin (Sigma) was added to the organ bath 30 minutes before the experiment.

Statistical Analysis
Data are expressed as mean±SEM. Differences were compared by paired Student t test or one-way repeated measures ANOVA followed by the Student-Newman-Keuls test when appropriate. Statistical significance was taken as P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inotropic Effects of IGF-1
IGF-1 (0.1 µmol/L) exerted pronounced positive inotropic effects in isolated human cardiac muscle (Figure 1A, top panel). The inotropic response developed to a stable plateau phase within 3 to 4 minutes without changes in diastolic tension. These functional effects of IGF-1 were mediated via specific IGF-1 receptors; preincubation of a preparation from the same heart with the specific IGF-1 receptor antibody IR-3 (3.3 µg/mL) for 30 minutes prevented the inotropic response to IGF-1 (bottom panel).

View larger version (16K): [in this window] [in a new window]   Figure 1. A, Top, Effect of 0.1 µmol/L IGF-1 on isometric twitch tension in a muscle strip from an end-stage failing human heart. Bottom, Same protocol after preincubation with IGF-1 receptor antibody IR3. B, Cumulative concentration-response curves for IGF-1 (0.001 to 0.2 µmol/L) in the absence (; n=11 preparations from 10 hearts) and presence ( n=7 preparations from 6 hearts) of IGF-1 receptor antibody IR3. *P<0.05 vs base.

The functional effects of IGF-1 were concentration dependent (Figure 1B). The inotropic response started at 0.01 µmol/L and was maximal at 0.1 µmol/L with an increase in twitch tension to 133.4±4.4% of the baseline value (n=11, P<0.05). The EC₅₀ was 0.020 µmol/L (95% confidence interval = 0.015 to 0.027 µmol/L). Diastolic tension and time parameters of contraction and relaxation were not affected by IGF-1. There were no significant differences in inotropic responses to IGF-1 between trabeculae from hearts with dilated or ischemic cardiomyopathy. As can also be seen from Figure 1B, preincubation of muscle strips with the IGF-1 receptor antibody IR-3 (n=7) almost completely prevented the inotropic response to IGF-1.

Subcellular Mechanisms of Action of IGF-1
Further experiments were performed in aequorin-loaded muscle strips and compared with the effects of increasing [Ca²⁺]_o from 2.5 to 4.0 mmol/L or of ß-adrenoceptor stimulation with ISO (0.1 µmol/L). These concentrations were chosen to achieve inotropic effects comparable to those with IGF-1. Figure 2 shows original tracings of the effects of IGF-1 (0.1 µmol/L, left), and [Ca²⁺] (4.0 mmol/L, right) on aequorin signals (upper tracings) and the corresponding isometric twitches (lower tracings). IGF-1 resulted in a similar increase in the amplitude of the aequorin light signal and isometric twitch tension. Likewise, [Ca²⁺]_o 4.0 mmol/L increased both aequorin light emission and force of contraction to a similar extent. As can be seen from Figure 3 (left), IGF-1 (0.1 µmol/L, n=6) resulted in a proportional increase in twitch tension and aequorin light emission (by 46±9% and 51±12% of the basal value, respectively; P<0.05). To further elucidate the IGF-1-dependent signal transduction pathways, wortmannin (0.1 µmol/L) was used to block the PI3-kinase in aequorin-loaded muscle strips. Preincubation with wortmannin did not affect basal contractile force or relaxation (Table 1), but almost completely prevented both the increase in twitch force and Ca²⁺ transients after IGF-1 (Figure 3, right).

View larger version (26K): [in this window] [in a new window]   Figure 2. Superimposed original tracings of effects of IGF-1 (0.1 µmol/L, left) and [Ca2+]o (4.0 mmol/L, right) on intracellular Ca2+ transients (aequorin light signals, top) and isometric twitch tension (bottom).

View larger version (23K): [in this window] [in a new window]   Figure 3. Left, Effects of IGF-1 (0.1 µmol/L) on twitch force (filled bar) and aequorin light emission (open bar). Average data from 6 preparations from 6 hearts. Right, Same protocol as above, but after preincubation with 0.1 µmol/L wortmannin. Average data from 6 preparations from 6 hearts. Wortmannin did not affect basal force (-6.1±4.2%, NS) or Ca2+ transient amplitude (-10.2±7.4%, NS). *P<0.05 vs basal value before IGF-1 administration. % of base (ordinate) signifies percentage change from basal value of developed force or aequorin light emission before IGF-1 administration.

View this table: [in this window] [in a new window]   Table 1. Influence of Pharmacological Blockers on Basal Force and Relaxation

Inotropic effects may be brought about by cAMP-dependent or cAMP-independent mechanisms. cAMP-dependent interventions are usually associated with enhanced relaxation and an overproportional increase in intracellular Ca²⁺ transients.²³ Table 2 summarizes the effects of IGF-1, Ca²⁺, and ISO on twitch force, aequorin light emission, and twitch and Ca²⁺ transient decay parameters. All inotropic interventions yielded comparable inotropic responses. However, while the relative increase in force (F) and aequorin light emission (L) was similar with IGF-1 or [Ca²⁺]_o 4.0 mmol/L (F/L was 1.02±0.38 and 1.03±0.12, respectively), the increase in aequorin light emission relative to force was significantly higher with ISO (F/L was 0.56±0.11). Although this may indicate that ISO (in contrast to IGF-1) overproportionally increases intracellular Ca²⁺ transients relative to force, these relationships have been obtained only at one time point and must be interpreted with caution. Furthermore, relaxation time of the isometric twitch and decay time of the Ca²⁺ transient were significantly abbreviated with ISO but remained unchanged with IGF-1 and [Ca²⁺]_o 4.0 mmol/L (Table 2). These experiments suggest that IGF-1 exerts its positive inotropic effect by a cAMP-independent increase in intracellular Ca²⁺ transients.

View this table: [in this window] [in a new window]   Table 2. Influence of IGF-1, [Ca2+]o, and Isoproterenol on Force and Aequorin Light Amplitude and on Decay Time of Force and Aequorin Light

To support this hypothesis, we directly measured cAMP concentrations in trabeculae under control conditions and after stimulation with IGF-1 or ISO. Under control conditions, cAMP levels were 470±158 pg/mL and did not increase after preincubation with IGF-1 (616±126 pg/mL, NS). In contrast, after preincubation with ISO (0.1 µmol/L), cAMP concentrations were increased to 1208±247 pg/mL (P<0.05 versus control or IGF-1).

Effects of IGF-1 on SR Ca²⁺ Handling
We performed rapid-cooling experiments to directly assess the effects of IGF-1 on SR Ca²⁺ content. Figure 4A shows a representative original recording. The upper tracing reflects temperature near the surface of the muscle, and the lower tracing represents twitch force. At steady-state isometric contractions, the muscle was cooled to 1°C, and a stable cooling contracture as an index for SR Ca²⁺ content developed. On rewarming, the muscle completely relaxed, and the experimental protocol was repeated after addition of IGF-1. IGF-1 increased both isometric twitch tension and the amplitude of the cooling contracture. The average data are presented in Figure 4B (left). IGF-1 (0.1 µmol/L) significantly increased twitch force and RCCs by 38.8±9.3% and 27.6±9%, respectively. This indicates that the inotropic effect of IGF-1 is associated with increases in SR Ca²⁺ content. Then, we blocked SR Ca²⁺ storage function by preincubating muscle strips with cyclopiazonic acid (20 µmol/L) and ryanodine (1 µmol/L). This resulted in a significant decline in twitch force (by 42%) and prolongation of relaxation times (Table 1). However, no RCCs could be elicited under these conditions, and contractions may result exclusively from transsarcolemmal Ca²⁺ cycling.²⁰ Interestingly, the inotropic response to IGF-1 was unchanged with a blocked SR; as can be seen from Figure 4B (right), IGF-1 (0.1 µmol/L) increased force of contraction by 43.7±14.2% (P<0.05) without detectable RCCs in the presence of cyclopiazonic acid and ryanodine.

View larger version (21K): [in this window] [in a new window]   Figure 4. A, Original recording of rapid cooling experiment. Top part depicts bath temperature; bottom part, twitch force. Upon cooling, a stable cooling contracture develops. With rewarming, the muscle completely relaxes after a brief rewarming spike. At steady-state conditions, IGF-1 (0.1 µmol/L) is added, and the experiment is repeated. B, Left, Average values from 7 preparations from 7 hearts for experiments as shown in Figure 4A. Right, Additional experiments were performed in 7 preparations from 6 hearts after blocking SR function with cyclopiazonic acid (CPA) and ryanodine. Data are compared with basal values before IGF-1 administration. This intervention completely prevented RCCs but not the increase in twitch force with IGF-1. % of base (ordinate) signifies percentage change from the basal value of developed force or RCC amplitude before IGF-1 administration.

In additional experiments, we used paired cooling contractures to test whether IGF-1 directly affected SR Ca²⁺ uptake.²⁰ The rationale for these experiments is that during the rewarming period (in the unstimulated muscle), cytosolic Ca²⁺ is competitively removed from the cytosol by sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2a and Na⁺-Ca²⁺ exchange. The RCC2/RCC1 ratio in failing human trabeculae was 52% in the absence of and 51% in the presence of 0.1 µmol/L IGF-1. This indicates that 52% of the cytosolic Ca²⁺ is reaccumulated into the SR, whereas 48% is eliminated from the cytosol via Na⁺-Ca²⁺ exchange, and that this ratio is not affected by IGF-1.

Effects of IGF-1 on Transsarcolemmal Ca²⁺ Influx
We further assessed the contribution of transsarcolemmal Ca²⁺ influx to inotropic responses to IGF-1. Major routes of Ca²⁺ entry in human myocardium are through L-type Ca²⁺ channels or reverse-mode Na⁺-Ca²⁺ exchange.^21,24

We first tested whether IGF-1 affects transsarcolemmal Ca²⁺ influx through L-type Ca²⁺ channels. For this purpose, we analyzed L-type Ca²⁺ currents by whole-cell voltage-clamp techniques in isolated cardiomyocytes. Figure 5A shows typical current recordings during depolarization steps from -40 to +10 mV. It can be seen that the amplitude of the Ca²⁺ current increases, while the decay kinetics remain unchanged. Similar experiments were performed in a total of 5 ventricular myocytes (Figure 5B). In these experiments, the basal current amplitude induced by the voltage step from -40 to +10 mV was 2.54±0.63 nA. IGF-1 (0.1 µmol/L) significantly increased I_Ca,L amplitude to 124±7% of the basal value.

View larger version (20K): [in this window] [in a new window]   Figure 5. A, Original recording of L-type Ca2+ current in a human cardiac myocyte in the absence (left) and presence (right) of 0.1 µmol/L IGF-1. B, Average increase in L-type Ca2+ current amplitude. Experiments were performed in 5 myocytes from 3 failing hearts. CTRL indicates control.

In a separate set of experiments in trabeculae, the effects of preincubating muscle strips with the L-type Ca²⁺ channel antagonist diltiazem (5 µmol/L) on the inotropic response to IGF-1 was investigated. At this concentration, diltiazem exerted a pronounced negative inotropic effect (Table 1). Applied at contractile steady-state conditions, the inotropic response to IGF-1 was significantly reduced in the presence of the Ca²⁺ channel blocker (Figure 6). These experiments suggest that the increase in intracellular Ca²⁺ transients and twitch force with IGF-1 are partly mediated by enhanced Ca²⁺ entry through L-type Ca²⁺ channels.

View larger version (37K): [in this window] [in a new window]   Figure 6. Influence of diltiazem (Dilt; 5 µmol/L), HOE642 (HOE; 3 µmol/L), and KB-R7943 (KB-R; 5 µmol/L) on inotropic response to IGF-1. Experiments were performed in 8 preparations from 5 hearts, 6 preparations from 4 hearts, and 7 preparations from 5 hearts, respectively. IGF-1 was always added at steady-state contractile conditions. Inotropic response to IGF-1 without preincubation is shown for comparison (open bar). *P<0.05 vs IGF-1 effects without preincubation.

Intracellular Ca²⁺ accumulation may also be modulated by the Na⁺-Ca²⁺ exchanger. This electrogenic ion transporter extrudes Ca²⁺ for Na⁺ influx in its forward mode but may also work in its reverse mode, resulting in Ca²⁺ influx during depolarization.²⁴ The latter may be favored by increases in [Na⁺]_i. Interestingly, recent experiments demonstrated an IGF-1-mediated stimulation of the sarcolemmal Na⁺-H⁺ exchanger in vascular smooth muscle cells.²⁵ In the present study, preincubation of muscle strips with the Na⁺-H⁺ exchange inhibitor HOE642 (3 µmol/L) only slightly affected basal force (Table 1) but resulted in a significant reduction in the maximal inotropic response to IGF-1 (by 60±15%; P<0.05; Figure 6). In a further set of experiments, the effects of the reverse-mode Na⁺-Ca²⁺ exchange inhibitor KB-R7943 (5 µmol/L) were tested. KB-R7943 exerted a significant depressant effect on basal force of contraction (Table 1). Applied at steady-state contractile conditions, the positive inotropic effect of IGF-1 was reduced by 70±9% (P<0.05; Figure 6) in the presence of KB-R7943. These data indicate that the inotropic response to IGF-1 is related to enhanced transsarcolemmal Ca²⁺ entry through both L-type Ca²⁺ channels and reverse-mode Na⁺-Ca²⁺ exchange. The latter may be secondary to an IGF-1-mediated activation of the Na⁺-H⁺ exchanger with subsequent [Na⁺]_i accumulation. Because both Na⁺-H⁺ exchange and L-type Ca²⁺channels may be activated through protein kinase C (PKC),^26,27 we preincubated muscle preparations with the PKC inhibitor GF109203X (n=4). PKC inhibition did not affect basal contractility (Table 1), but it resulted in a significant reduction in the inotropic response to IGF-1 by 69±4% (P<0.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This is the first report on functional effects of IGF-1 in isolated human myocardium. The results show that (1) IGF-1 exerts a concentration-dependent positive inotropic effect that is almost completely prevented by blocking IGF-1 receptors or PI3-kinase, (2) the inotropic effect is related to a cAMP-independent increase in intracellular Ca²⁺ transients, and (3) IGF-1 increases L-type Ca²⁺ currents and activates Na⁺-H⁺ exchange and reverse-mode Na⁺-Ca²⁺ exchange.

Acute and Long-Term Effects of IGF-1 in the Cardiovascular System
IGF-1 exerts acute hemodynamic as well as long-term genomic effects in the cardiovascular system. Genomic effects include induction of muscle growth, hypertrophy, and antiapoptotic signaling.^1,2,4 Experimental and clinical studies indicate that long-term application of GH or IGF-1 may have beneficial effects in heart failure.^3,10,28

In addition, acute application of IGF-1 to human healthy volunteers or to patients with heart failure increases cardiac output,^12,13 but the underlying mechanisms for these hemodynamic effects remain unclear. Noninvasive studies demonstrated endothelium-dependent vasodilatory actions of IGF-1 in humans, which could account for improved hemodynamics on infusion of IGF-1.^14,15 IGF-1 exerted direct positive inotropic effects in isolated mammalian cardiac muscle,^16–18 and inotropic effects of IGF-1 could contribute to enhanced circulatory function in humans. However, no data on direct functional effects of IGF-1 in the human heart are available.

Direct Functional Effects of IGF-1 in Isolated Human Myocardium
We observed a 35% increase in isometric twitch force at 0.1 µmol/L IGF-1 without changes in relaxation parameters or diastolic function. This inotropic response to IGF-1 amounts to 30% to 40% of the maximal positive inotropic effects of ß-adrenoceptor stimulation in failing human myocardium.²⁹ Therefore, the acute functional effects of IGF-1 are substantial and of potential clinical relevance. The magnitude of the inotropic responses to IGF-1 in this study is comparable to the effects previously reported in isolated neonatal¹⁶ and adult³⁰ rat myocytes, canine myocytes,¹⁸ and ferret trabeculae.¹⁷

The inotropic response to IGF-1 in the present study started at a concentration of 0.01 µmol/L (75.8 ng/mL) and was maximal at a concentration of 0.1 µmol/L (758 ng/mL). These concentrations are identical to those in isolated ferret muscle¹⁷ and comparable to experiments in isolated rat and dog myocytes.^19,29 In humans, IGF-1 plasma levels do not reach such high values.³¹ However, increases in local cardiac IGF-1 peptide content and upregulation of IGF-1 receptors as well as increased local cardiac IGF-1 release have been observed in myocardium from animal models of pressure or volume overload or ischemic cardiomyopathy.^5,7 In addition, myocardial hypertrophy was associated with a cardiac-specific increase in IGF-1 mRNA expression and peptide generation.⁶ Therefore, IGF-1 exerts its effects on cardiac myocytes not only in an endocrine but also an autocrine/paracrine fashion within the heart, and IGF-1 concentrations at the level of the myocytes may be substantially higher than in the systemic circulation.

Signal Transduction Pathways of IGF-1 in Human Myocardium
IGF-1 may bind to both types of IGF receptors and to insulin receptors, but the IGF-1 receptor, a heterotetrameric protein with intrinsic tyrosine kinase activity, is most abundant in adult myocardium.^31,32 In the present study, IGF-1 effects were almost completely prevented by preincubation with the selective IGF-1 receptor antibody IR-3. This demonstrates the role of specific IGF receptors of the subtype 1 in the functional effects of IGF-1 in human myocardium.

Activation of several signal transduction pathways after IGF-1 receptor stimulation were described.³³ One of these pathways involves phosphorylation of insulin-receptor substrate with consecutive activation of PI3-kinase.¹ The PI3-kinase-dependent pathway mediates antiapoptotic signaling and trophic effects via activation of Akt, a serine/threonine protein kinase,³⁴ and promotes glycogen synthesis through phosphorylation of glycogen synthase kinase 3.³⁴ In the present study, preincubation of human ventricular muscle with the selective PI3-kinase inhibitor wortmannin prevented the IGF-1-dependent inotropic effect and the increase in Ca²⁺ transients. This demonstrates that PI3-kinase activation is a key event in the signal transduction network ultimately resulting in functional effects. The involvement of PI3-kinase activation in the inotropic response to IGF-1 is consistent with a previous report in rat heart.¹⁸ On the other hand, PI3-kinase inhibition was described to modify ß₂-adrenoceptor signaling in adult rat myocardium enabling ß₂-adrenoceptors to induce phospholamban phosphorylation and increase contractility and Ca²⁺ transients.³⁵ This finding may point to distinct pathways of PI3-kinase signaling depending on the trigger and the consecutive activation of PI3-kinase isoforms.

In addition, PKC inhibition partly prevented the inotropic response to IGF-1 in the present study, and PKC activation occurred downstream from PI3-kinase activation in mammalian myocardium.³⁶ However, the exact signaling cascade responsible for functional effects of IGF-1 remains to be elucidated. For example, PI3-kinase-dependent Akt activation is a key element in the protection of cardiomyocytes from cell death,³³ but the contribution of Akt to functional effects of IGF-1 is unclear.

Influence of IGF-1 on Intracellular Ca²⁺ Handling
The functional responses to IGF-1 were accompanied by an increase in intracellular Ca²⁺ transients in the present study. An increase in [Ca²⁺]_i as the underlying mechanism for the inotropic effect of IGF-1 was also demonstrated in rat myocytes^18,30 but not in rat whole-heart preparations.¹⁷ The mechanisms involved in the increase in Ca²⁺ on acute administration of IGF-1 are unknown. Principally, a rise in [Ca²⁺]_i may result from cAMP-dependent (eg, ß-adrenoceptor stimulation) or cAMP-independent mechanisms.^23,29 The present data strongly suggest that the functional effects of IGF-1 result from cAMP-independent mechanisms for several reasons, as follows: (1) ß-adrenoceptor stimulation with ISO, but not IGF-1, enhanced relaxation rate and decay of the Ca²⁺ transients; (2) IGF-1 increased force and aequorin light emission similar to [Ca²⁺]_o, whereas the increase in aequorin light emission was much more pronounced with ISO; and (3) cAMP levels in isolated human myocardium did not change with IGF-1 but were largely increased with ISO.

Inotropic compounds may also directly modulate the responsiveness of the myofilaments for Ca²⁺. Previous reports on the effects of IGF-1 on myofilament Ca²⁺ sensitivity in mammalian myocardium are contradictory. Cittadini et al¹⁷ reported an IGF-1-dependent increase in myofilament Ca²⁺ sensitivity, but Kinugawa et al¹⁸ could not detect changes in the myofilament responsiveness in rat myocytes. A Ca²⁺-sensitizing mode of action typically results in little or no increase in intracellular Ca²⁺ transients, prolonged relaxation, and diastolic dysfunction.^23,29 Given that Ca²⁺ transients increased, and relaxation time as well as diastolic function remained unchanged in the present study, sensitization of the myofilaments does not appear to be a major mode of action of IGF-1 in human myocardium.

We extended the study of intracellular Ca²⁺ handling and assessed the effects of IGF-1 on SR function and Ca²⁺ content. RCCs revealed that the inotropic response to IGF-1 was associated with increased SR Ca²⁺ content. However, pharmacological blockade of SR function did not prevent the inotropic response to IGF-1. Therefore, the increase in SR Ca²⁺ load after IGF-1 stimulation probably results from increased Ca²⁺ availability within the myocytes and not from direct IGF-1 stimulation of SR Ca²⁺ uptake. This is also supported by the unchanged RCC2/RCC1 ratio in the present study.

Influence of IGF-1 on Transsarcolemmal Ca²⁺ Influx
Recently, Solem and Thomas³⁷ demonstrated a substantial increase in dihydropyridine-sensitive Ca²⁺ channel activity in cardiac myocytes on exposure to IGF-1. In fact, in the present study, L-type Ca²⁺ currents significantly increased in the presence of IGF-1 in isolated human cardiomyocytes. Furthermore, diltiazem partly prevented the inotropic response to IGF-1. In addition to L-type Ca²⁺ channel-mediated effects, inotropic responses to IGF-1 could be reduced to a similar extent by inhibition of the Na⁺-H⁺ exchanger with HOE642 or by the reverse mode of the Na⁺-Ca²⁺ exchanger with KB-R7943. Therefore, activation of Na⁺-H⁺ exchange contributes to the acute effects of IGF-1 in human myocardium, possibly via enhanced Ca²⁺ entry through [Na⁺]_i-dependent activation of the reverse-mode Na⁺-Ca²⁺ exchange function.²⁴ This is supported by a recent study in rat smooth muscle cells in which IGF-1 increased [Ca²⁺]_i through activation of Na⁺-H⁺ exchange.²⁵

Taken together, in myocardium from failing human hearts, the functional effects of IGF-1 are mediated by PI3-kinase-dependent increases in intracellular Ca²⁺ transients. The data indicate that IGF-1-induced activation of L-type Ca²⁺ channels, as well as Na⁺-H⁺ exchange, followed by enhanced reverse-mode Na⁺-Ca²⁺ exchange, contributes to the increase in force and [Ca²⁺]_i. Neither increases in cAMP nor altered Ca²⁺ responsiveness of the myofilaments appears to contribute significantly to the acute inotropic responses to IGF-1. Our data indicate that the potential pathophysiological and clinical relevance of IGF-1-dependent signaling in human heart failure deserves further investigation.

Limitations of the Study
One limitation of the study is that isolated muscle strip preparations produce less contractile force than could be expected from the undamaged, intact organ. However, the basal developed force of the muscles used in this study (22±2 mN/mm²) is comparable or even higher as reported in previous studies using failing human myocardium.^19,20,38 Nevertheless, muscle strips may undergo damage during transport and preparation, and this may affect the results. A further limitation of the study is that aequorin light signals have not been converted to [Ca²⁺]_i. This in itself is not a problem, but it largely precludes direct comparison between different inotropic interventions. Although validated for human cardiac muscle,²⁰ rapid cooling may not release all Ca²⁺ stored within the SR.³⁹ In addition, RCCs are an indirect measure of SR Ca²⁺ content, and subcellular changes, such as a potential IGF-1-induced increase in myofilament responsiveness to Ca²⁺, may also result in increased RCCs. The postulated IGF-1-dependent activation of Na⁺-H⁺ exchange could result in increased pH_i resulting in enhanced myofilament Ca²⁺ responsiveness. However, changes in pH_i are unlikely to occur in the presence of bicarbonate-containing physiological buffer solutions⁴⁰ and could not be detected in preliminary (n=3) experiments using BCECF in human preparations. Furthermore, pharmacological blockade of Na⁺-H⁺ exchanger 1 and, more likely, reverse-mode Na⁺-Ca²⁺ exchange may not be fully specific.^41,42 Therefore, these experiments indicate, but do not prove, involvement of sodium-dependent exchangers in the inotropic response to IGF-1.

	Acknowledgments

 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft to B.P. (DFG Pi-414/1).

Received August 29, 2001; revision received July 31, 2002; accepted December 3, 2002.

	References

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