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Cardiovascular Research 1999 42(1):113-120; doi:10.1016/S0008-6363(98)00307-1
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Copyright © 1999, European Society of Cardiology

Insulin stimulates the L-type Ca2+ current in rat cardiac myocytes

Friedbert Aulbacha, Andreas Simmb, Sebastian Maiera, Heiner Langenfelda, Ulrich Waltera, Ulrich Kerstinga and Michael Kirsteina,*

aMedical University Clinic, Institute of Clinical Biochemistry and Pathobiochemistry, 97080 Würzburg, Germany
bPhysiological Chemistry II, Theodor-Boveri-Institut für Biowissenschaften, 97074 Würzburg, Germany

* Corresponding author. Tel.: +49-931-201-2775; fax: +49-931-201-2775; e-mail: m.kirstein@medizin.uni-wuerzburg.de

Received 29 June 1998; accepted 19 September 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The aim was to study the L-type calcium current (ICa,L) in cardiac myocytes as a possible target of insulin in the regulation of cardiac function. Method: Using the whole-cell configuration of the patch–clamp technique, we investigated the stimulation of ICa,L by insulin in isolated rat ventricular myocytes. Results: The stimulation of ICa,L by insulin was dose-dependent (EC50=33 nM) and reversible. Maximum stimulation of ICa,L over basal ICa,L was 86±11% (n=25) at 1 µM insulin. Insulin (1 µM) shifted the current–voltage relationship and potential-dependent availability of ICa,L to more negative potentials by about 3.5 and 1.5 mV, respectively. The maximum conductance of ICa,L was increased by 1 µM insulin, from 26±4 to 39±5 nS (n=11). Isoproterenol (100 nM), which stimulated ICa,L by 156±23% (n=10) over basal ICa,L, acted faster than insulin. The half-maximum stimulation of ICa,L by isoproterenol and insulin was reached after 44±5 and 80±9 s, respectively. Insulin and isoproterenol responses were not additive. Insulin (1 µM) and isoproterenol (100 nM) stimulation of ICa,L was inhibited by Rp-cAMPS (1 mM) to 12±3 and 32±4%, respectively. Insulin (1 µM) increased cAMP content in rat cardiomyocytes by about two-fold. Insulin-like growth factor-1 (IGF-1; 5 µM) increased ICa,L by only 5.9±0.9% (n=6). Conclusions: Our data show that insulin stimulates the L-type calcium current in isolated rat ventricular myocytes in a dose-dependent and reversible manner and suggest that this effect is mediated by insulin receptors and the cAMP-dependent protein kinase.

KEYWORDS Calcium current, L-type; Rat, ventricular myocytes; Patch–clamp; cAMP


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Insulin is a crucial regulator of metabolic physiology, blood glucose homeostasis, cellular glucose and amino acid uptake. Insulin also regulates gene expression, ion flux, cell proliferation and apoptosis [1]. In mammalian heart and in isolated cardiac muscle preparations, insulin exerts positive inotropic effects [2–7]. These cardiac effects include increases in maximum force generation, left ventricular dP/dtmax, cardiac output and faster relaxation times. Positive inotropic effects of insulin are independent of the myocardial glycolytic rate and substrate metabolism [2–5]and independent of adrenergic mechanisms [2, 3, 6, 7]. Positive inotropic effects of catecholamines have been well investigated. The crucial event in the catecholamine-stimulated signal-event cascade in the heart is an increased Ca2+ influx mediated by cAMP-dependent protein kinase (PKA) [8–11].

Previous studies have suggested that insulin may alter cation fluxes in the myocardium, particularly that of Ca2+, and thereby may change mechanical force development in the heart [12–14]. Relaxin, a reproductive hormone of the insulin family, increased heart rate by stimulating the L-type Ca2+ current (ICa,L) in rabbit sino-atrial node cells [15]. Insulin stimulated Ca2+ ATPase activity in both the sarcoplasmic reticulum and the sarcolemma and increased the Na+/Ca2+ exchange activity [12, 14, 16]. Insulin-stimulated Ca2+ uptake is involved in the regulation of heart metabolism and transporter activities [14, 17]. In order to study a possible insulin-induced Ca2+ influx in cardiac cells, we applied the whole-cell mode of the patch–clamp technique. Our data demonstrate that the regulation of cardiac ICa,L is a new mechanism of the action of insulin.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell isolation
Single rat cardiac myocytes were prepared according to the protocol of Piper et al. [18]with minor modifications. The resulting rod-shaped myocytes were used within 24 h. Overnight storage did not significantly change the maximum stimulation of ICa,L by insulin. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.

2.2 Chemicals
Insulin (from bovine pancreas), (–)-isoproterenol and insulin-like growth factor-1 (IGF-1; human, recombinant analog) were purchased from Sigma (Deisenhofen, Germany). IGF-1 was prepared as a 25 µM stock solution [containing 800 mg/l bovine serum albumin (BSA) to increase stability of IGF-1], stored at –20°C, and diluted with an external solution to the final concentrations less than 15 min before being applied to the cells. Insulin and isoproterenol were dissolved in external solution directly before use. Rp-cAMPS was purchased from BIOLOG (Bremen, Germany). All other chemicals were of analytical grade and were obtained from Merck (Darmstadt, Germany).

2.3 Electrophysiological recordings
Membrane currents were measured at room temperature (19–23°C) using the whole-cell configuration of the patch–clamp technique [19]. Myocytes were allowed to settle for 5 min before being superfused with external solution at a rate of about 1 ml/min. The control superfusion solution (external) and the pipette filling solution (internal) were designed to block K+ currents. External solution contained 107.1 mM NaCl, 30 mM CsCl, 1.8 mM MgCl2, 1.8 mM CaCl2, 4 mM NaHCO3, 5 mM glucose, 5 mM sodium pyruvate, 10 mM HEPES and 0.8 mM NaH2PO4·2H2O, adjusted to pH 7.4 with NaOH. The internal solution was 119.8 mM CsCl, 4 mM MgCl2, 5 mM creatine phosphate disodium salt, 3.1 mM Na2ATP, 0.42 mM NaGTP, 5 mM EGTA, 62 µM CaCl2 (pCa 8.5) and 10 mM HEPES, adjusted to pH 7.2 with CsOH.

Patch pipettes were fabricated from borosilicate glass capillaries (KIMAX-51, Witz-Scientific, Holland, OH, USA) with resistances of 0.5–1.5 M{Omega}. After obtaining a gigaseal, a suction pulse combined with an electrical pulse (1 V, 5 ms) was applied to establish the whole-cell mode. Sealed ventricular myocytes were brought in front of the opening of a microcapillary. Microcapillaries were perfused by external solution at a rate of about 10 µl/min, corresponding to a flow velocity of 0.4 mm/s. Three additional microcapillaries, mounted in a row, allowed superfusion solutions to be changed within 4 s by moving the capillaries.

Command pulses and data acquisition were performed with an EPC-9 patch–clamp amplifier controlled by the ‘PULSE’ software (HEKA, Lambrecht, Germany). In order to examine ICa,L, cells were voltage-clamped at a holding potential of –50 mV, to inactivate the sodium current, and 200 ms depolarizing pulses to 0 mV were applied at 0.1 Hz [20]. For the determination of current–voltage relationships of ICa,L (see Fig. 2A) and of ICa,L inactivation curves (see Fig. 2B), a double-pulse voltage-clamp protocol was used [21, 22](see inset in Fig. 2B).


Figure 2
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  		Fig. 2 Effects of insulin on the voltage-dependent characteristics of ICa,L. The protocol used in rat ventricular myocytes to construct current–voltage relations (A), activation curves (d{infty}) and availability curves (f{infty}) (B) is shown in the inset of (B). Conditioning-pulse (CP) potentials were separated from the test pulse (TP) by a 3 ms-return to –50 mV. Pulse pairs were applied every 2 s. A single experiment is shown in (A) and (B). (A) For construction of the current–voltage relation, ICa,L during CP was plotted against CP potentials. (B) To construct the availability curves, ICa,L during TP was normalized to the current obtained at the holding potential and plotted as a function of the CP potential.

 
For characterization of the activation (current–voltage relation), the method described by Isenberg and Klöckner [23]was used. Briefly summarized, the apparent reversal potential, ECa, and the maximum conductance were estimated by extrapolation from the steep linear ascending part of the I–V relation to the zero-current axis and its slope. The peak conductance (gCa,L) for each membrane potential (V) was then calculated with the following equation: gCa,L=ICa,L/(V–ECa). The peak conductances were normalized to the maximum conductance and the resulting curve was fitted by a Boltzmann distribution, d{infty}=1/{1+exp[(V1/2–V)/k]}, where d{infty} is the normalized conductance, V1/2 is the membrane potential at which the conductance is activated at its half-maximum value, and k is the slope factor. The potential of the half-maximum available ICa,L was determined by linear interpolation of the availability curve.

2.4 Determination of cAMP
Cyclic AMP in rat cardiomyocytes was determined by radioimmunoassay (RIA). We used a commercial RIA kit (RPA 509, Amersham International, UK) and the non-acetylated protocol described in the manufacturer’s instructions. Cyclic AMP was extracted from rat cardiomyocytes by the liquid phase extraction method for cell suspensions, as described in the manufacturer’s instructions. Cell number and mean single cell volume (20.6±2.7 pl) of rat cardiomyocytes was determined with a coulter-like cell counter Casy 1 system (Scherfe, Reutlingen, Germany). The single cell volume of cardiomyocytes was used to estimate basal intracellular cAMP concentration. Cells were preincubated for 5 min with the inhibitor of cAMP phosphodiesterase, IBMX (100 µM), to prevent the rapid degradation of the agonist-induced increased cAMP [24–26]. Insulin (1 µM) and isoproterenol (100 nM) were added for 2 min.

2.5 Data analysis and statistics
ICa,L was measured on-line as the difference between the peak inward current and the average current during the last 10 ms of the 200 ms pulse. This end-pulse current was small compared to ICa,L and not affected significantly by insulin. Cell membrane capacitance and series resistance (RS) were measured but not compensated for by exponential analysis of current responses to 5 mV pulses at regular intervals. Only measurements with a stable RS≤6 M{Omega} were analyzed. Membrane capacitance was 192±7 pF (mean±SEM) and RS was 4.3±0.2 M{Omega} (n=66). For off-line data analysis, IGOR software from WaveMetrics (Lake Oswego, Oregon, USA) was used.

Results are presented as mean±SEM. Differences between means were tested for statistical significance by Student’s t-test for paired or unpaired samples, as necessary. Differences were considered significant at the level of p<0.05 or less, as indicated.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
ICa,L was recorded in rat ventricular myocytes using the whole-cell configuration of the patch–clamp technique. Usually 8–10 min were allowed for stabilization of rundown of ICa,L before measurements. Basal ICa,L activated by a voltage step from –50 to 0 mV was 1200±89 pA (n=66). The ICa,L density, which represents the ratio of ICa,L amplitude to the membrane capacitance, was 5.9±0.3 pA/pF (n=66).

3.1 Insulin stimulation of ICa,L
Fig. 1 shows a representative example of the stimulatory effect of 1 µM insulin on ICa,L. Washout of insulin reduced ICa,L to its basal amplitude, demonstrating that the effect was fully reversible. On averaging the results of 25 experiments, 1 µM insulin increased ICa,L by 86±11% (p<0.001) over basal. The maximum insulin effect was obtained after 185±13 s and the half-maximum effect was reached after 80±9 s (n=13). The time to peak was shortened from 6.9±0.2 (basal) to 6.0±0.3 ms by 1 µM insulin (n=13, p<0.001). The current–voltage relation, activation and inactivation voltage-dependencies of ICa,L are shown in Fig. 2. The current–voltage relation was shifted to more negative potentials by insulin (V1/2=–2.4±0.8 mV (basal) vs. –6.0±0.9 mV (insulin), n=11, p<0.001) whereas the slope factor of the activation curve was not affected (6.9±0.2 mV/e-fold). The apparent reversal potential of ICa,L was not altered by insulin [56.2±0.9 mV (basal) vs. 55.8±1.0 mV (insulin)]. The maximum conductance gmax was increased from 26.2±3.8 nS (basal) to 39.2±5.0 nS by insulin (n=11, p<0.001). The availability curve was shifted slightly to more hyperpolarized potentials [half-maximum at –24.8±0.9 mV (basal) vs. –26.3±0.9 mV (insulin), n=11, p<0.01].


Figure 1
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  		Fig. 1 Stimulation of ICa,L by insulin. ICa,L was activated in rat ventricular myocytes by test pulses to 0 mV from a holding potential of –50 mV every 10 s (filled circles). External solution was switched to an insulin (1 µM)-containing solution, indicated by horizontal bars. Current traces before (a) and after (b) the addition of 1 µM insulin are shown in the inset. Cell capacitance was 187 pF and RS was 5.2 M{Omega} in the experiment shown.

 
3.2 Dose-dependency of insulin-induced increase of ICa,L
The insulin-induced increase of ICa,L was dose-dependent (Fig. 3). The threshold of the insulin-induced increase of ICa,L was 1 nM (5±2%, n=15, p<0.01), and the maximum increase of ICa,L was obtained with 1 µM insulin. A cumulative dose of 10 µM insulin at 1 µM insulin did not further stimulate ICa,L (n=3, data not shown). The EC50 of the dose–response curve was 33 nM and Emax was 90% over basal ICa,L.


Figure 3
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  		Fig. 3 Dose–response curve of the insulin-induced increase of ICa,L in rat ventricular myocytes. Increase of ICa,L over basal current is shown in percent. The dose–response relation between the insulin concentration and the increase in ICa,L amplitude was fitted to the Michaelis–Menten equation. The numerical values derived for Emax and EC50 were 90% and 33 nM, respectively. Each symbol represents the mean±SEM, with the number of tested cells indicated in parentheses.

 
3.3 Comparison of insulin- and isoproterenol-induced increases of ICa,L
At a concentration of 100 nM, isoproterenol increased ICa,L by 156±23% (n=10, p<0.001) over basal values (Fig. 4). Half of the maximum effect was reached after 44±5 s (n=10) with isoproterenol (100 nM) as compared to 80±9 s (n=13) with 1 µM insulin (p<0.005).


Figure 4
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  		Fig. 4 Time course of insulin and isoproterenol stimulation of ICa,L. ICa,L was activated in rat ventricular myocytes by test pulses to 0 mV from a holding potential of –50 mV every 10 s (filled circles). External solution was switched to an insulin- (1 µM) or isoproterenol (100 nM)-containing solution, indicated by horizontal bars. Current traces before (a) and after (b) the addition of 1 µM insulin, and after the addition of 100 nM isoproterenol (c) are shown in the inset.

 
3.4 Effect of IGF-1 on ICa,L
To examine the possible involvement of IGF-1 receptors in the observed increase in ICa,L by insulin, we applied IGF-1 to isolated rat cardiomyocytes. At 100 nM IGF-1, which was around three times the EC50 of insulin, there was no effect on ICa,L. In six experiments with 5 µM IGF-1, we found a small increase in ICa,L, by 5.9±0.9% over the basal current (p<0.005). Since 5 µM IGF-1 was dissolved in a solution containing 160 mg/l BSA, we tested a possible stimulation of ICa,L by BSA. We found an instantaneous increase in ICa,L (2.6±1.0%, n=4) by application of 160 mg/l BSA, which was not significant compared to the basal current, possibly due to the small number of experiments carried out. Compared to BSA-stimulated ICa,L, the increase in ICa,L by 5 µM IGF-1 was significant.

3.5 Involvement of cAMP/PKA pathway
Isoproterenol stimulated ICa,L in rat cardiomyocytes by activation of PKA, and insulin is known to stimulate adenylyl cyclase in mollusc and to increase intracellular cAMP in smooth muscle cells. To examine a possible involvement of the cAMP/PKA pathway in the insulin response in rat cardiomyocytes, we looked for possible additive or synergistic mechanisms of insulin and isoproterenol responses. Insulin (1 µM) failed to further stimulate ICa,L (1±1% increase in ICa,L by insulin over isoproterenol-stimulated ICa,L) when added to an isoproterenol (100 nM)-containing perfusion solution (n=3). The addition of isoproterenol to an insulin-containing solution induced a variable response. Isoproterenol (100 nM) further stimulated ICa,L (82% increase in ICa,L by isoproterenol over insulin-stimulated ICa,L) when added to an insulin (1 µM)-containing perfusion solution in an experiment with a relatively low insulin response (22% increase in ICa,L by insulin over basal Ca2+ current). Isoproterenol only slightly stimulated ICa,L further (12% increase in ICa,L by isoproterenol over insulin-stimulated ICa,L) when added to insulin (1 µM)-containing perfusion solution in an experiment with a relatively high insulin response (182% increase in ICa,L by insulin over basal Ca2+ current). When we stimulated ICa,L with 50 µM IBMX (108±22%, n=3, p<0.05 vs. basal), insulin was not able to increase ICa,L further. These experiments indicate converging intracellular signalling pathways of isoproterenol- and insulin-stimulated responses.

We then inhibited PKA by the addition of 1 mM Rp-cAMPS [27]to the internal pipette solution [8], which was without effect on basal ICa,L. Isoproterenol (100 nM) and insulin (1 µM) responses were measured about 20 min after break of the patch. Stimulation of ICa,L by isoproterenol without Rp-cAMPS was 147±32% (n=6) over basal current. In the presence of Rp-cAMPS, stimulation of ICa,L by isoproterenol was significantly reduced, to 32±4% (n=6, p<0.01) over basal current. Stimulation of ICa,L by insulin without Rp-cAMPS was 65±14% (n=6) over basal current. In the presence of Rp-cAMPS, stimulation of ICa,L by insulin was significantly reduced, to 12±3% (n=6, p<0.01) over basal current.

To further prove a possible involvement of the cAMP/PKA pathway in the insulin response, we measured the intracellular content of cAMP in isolated rat cardiomyocytes. The basal content of cAMP in rat cardiomyocytes was 1.04±0.13 pmol/105 cells (n=8). IBMX raised the cAMP content to 1.83±0.35 pmol/105 cells (n=7). As a positive control, we confirmed as a first step the well known increase in cAMP by isoproterenol in rat heart, which was 3.79±0.61 pmol/105 cells (n=9, p<0.001 vs. IBMX alone) in our study. Insulin increased intracellular cAMP content up to 3.69±0.48 pmol/105 cells (n=8, p<0.001 vs. IBMX alone), which was comparable to the increase induced by isoproterenol.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The data described in our study demonstrate for the first time that insulin stimulates ICa,L in mammalian cardiac cells. From a holding potential of –50 mV, where sodium currents are inactivated, we applied test pulses to 0 mV to selectively activate ICa,L. Sodium currents could be stimulated when the holding potential was decreased to –70 mV. Potassium currents were blocked by cesium ions added to the external and internal solutions.

Stimulation of the ICa,L by insulin in our study was dose-dependent and reversible. Most of the observed current increase was due to an increase in the maximum conductance. A minor part can be explained by changes in voltage-dependent parameters. Whereas the shift of the voltage-dependent availability was without effect on current amplitude, the shift of the activation curve lead to larger currents. Indeed, under basal conditions, the ICa,L was activated to 59% with a pulse to 0 mV. In contrast, the shift due to insulin lead to an activation of 71% with the same pulse amplitude.

Activation or inhibition by insulin of different types of Ca2+ channels that were not characterized as L-type Ca2+ channels have been observed in frog atrial cells [28]. In embryonic chick hearts, insulin failed to activate ICa,L in single ventricular myocytes [29]. Species differences, other insulin concentrations, and various differentiation states of the cardiac cells used may explain the contrasting results of Bkaily et al. [29]and our present results. Furthermore, positive controls of insulin effects on heart and the well known activation of the cardiac ICa,L by isoproterenol [8, 30]were not shown in the study by Bkaily et al. [29]. In vascular smooth muscle cells, insulin decreases ICa,L and contributes to insulin-induced vasodilation [31].

In our study, the insulin concentrations required to induce the half-maximal increase of ICa,L (33 nM or 5.4 mU/ml) were in the same range as used in the majority of previously published in vitro and in vivo studies reporting effects on cardiac contractility or intracellular calcium [2, 3, 14]. Insulin concentrations observed in blood are between 0.3 and 3 nM. However, heart has been reported to contain a hundredfold higher insulin concentration than blood [32], indicating that the concentrations of insulin used in our and other studies are physiologically relevant.

Presently, we only have limited information on the signalling pathway of ICa,L stimulation by insulin in cardiac myocytes. Insulin and isoproterenol or insulin and IBMX responses were not additive, indicating converging signalling pathways of both agonists in isolated cardiomyocytes. Furthermore, inhibition of isoproterenol- and insulin-stimulated ICa,L by Rp-cAMPS indicates that PKA is involved in both signalling pathways. Finally, insulin increases cAMP in rat cardiomyocytes. The estimated value of basal cAMP concentration in isolated rat cardiomyocytes in our study was 0.5 µM, which is in the same range as has previously been published for rat heart [33, 34].

Interestingly, insulin increased the cAMP and cGMP content in human vascular smooth muscle cells [25], and insulin and EGF activate the adenylyl cyclase system in a bivalve mollusc [35]. In rat heart, EGF stimulated cAMP accumulation [36]and activated the adenylyl cyclase system [26].

We confirmed other studies [37]that a maximum stimulation of ICa,L was obtained at 100 nM isoproterenol. Stimulation of ICa,L by isoproterenol in our study was faster and more sensitive in comparison to insulin. In contrast, isoproterenol and insulin increased cAMP similarly. One possible explanation is that cAMP measurements detect the total changes in a cell whereas the increase in ICa,L may depend on the compartmentation of the cAMP/PKA system to the calcium channels. Other features of ICa,L in our study, such as the insulin-induced increase in maximum conductance and the small shift in potential-dependent parameters, were similar to the effects of isoproterenol reported by others [10].

Both IGF-1 [38]and insulin [12, 16, 39]receptors have been detected in mammalian hearts, and stimulation of the IGF-1 receptor by insulin has been reported and vice versa [40, 41]. However, the KD values for both drugs differ by around two–three orders of magnitude. Therefore, if insulin stimulates ICa,L via IGF-1 receptors, IGF-1 should be two–three orders of magnitude more potent in stimulating ICa,L. The opposite was the case in our study. Even at a high concentration of IGF-1 (5 µM), stimulation of ICa,L was tiny, about 7% of the maximum effect of insulin at 1 µM. In order to ascertain the biological potency of our IGF-1 preparation, the known stimulating effect of IGF-1 on myocardial contractility [42, 43]was examined. Indeed, our IGF-1 solution (100 nM) increased the force of contraction in an isovolumic Langendorff heart by 40% (H. Strömer, unpublished data, details of method described by Strömer et al. [42]). Therefore, it is unlikely that insulin stimulates cardiac ICa,L by binding to IGF-1 receptors.

A diabetic cardiomyopathy has been shown in patients with normal coronary arteries and without atherosclerosis or hypertension [44–48]. There is convincing evidence from studies at the cellular level that, in diabetes, an abnormal intracellular handling of Ca2+ contributes to diabetic cardiomyopathy [13, 44, 45, 47–50]. Isolated myocytes from diabetic rats show contractile dysfunction that depends on changes in Ca2+ regulation [51]. Insulin treatment completely reverses or prevents cardiomyopathy in diabetic rats [52]. Altered cardiac Ca2+ metabolism may therefore play a role in metabolic disorders associated with hyperinsulinemia, insulin resistance and hypertension [48, 53, 54]. Future investigations will hopefully unravel the mechanism of L-type calcium channel stimulation by insulin and its role in cardiac physiology and pathophysiology.

Time for primary review 28 days.


   Acknowledgements
 
We thank Dr. H. Strömer for performing the measurements of positive inotropic effects of IGF-1 on the isolated rat heart and A. Katzer for her excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 355/TP B3, TP C2, and Ki 606/1-1).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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