Insulin and Inositol 1,4,5-Trisphosphate Trigger Abnormal Cytosolic Ca2+ Transients and Reveal Mitochondrial Ca2+ Handling Defects in Cardio
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糖尿病学杂志 2005年第8期
Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
ABSTRACT
Obesity, insulin resistance, and type 2 diabetes are leading causes of heart failure, and defective cellular Ca2+ handling seems to be a fundamental problem in diabetes. Therefore, we studied the effect of insulin on Ca2+ homeostasis in normal, freshly isolated mouse ventricular cardiomyocytes and whether Ca2+ handling was changed in an animal model of obesity and type 2 diabetes, ob/ob mice. Electrically evoked Ca2+ transients were smaller and slower in ob/ob compared with wild-type cardiomyocytes. Application of insulin (6 or 60 nmol/l) increased the amplitude of Ca2+ transients in wild-type cells by 30%, whereas it broadened the transients and triggered extra Ca2+ transients in ob/ob cells. The effects of insulin in ob/ob cells could be reproduced by application of a membrane-permeant inositol trisphosphate (IP3) analog and blocked by a frequently used IP3 receptor inhibitor, 2-aminoethoxydiphenyl borate. In ob/ob cardiomyocytes, insulin increased the IP3 concentration and mitochondrial Ca2+ handling was impaired. In conclusion, we propose a model where insulin increases IP3 in ob/ob cardiomyocytes, which prolongs the electrically evoked Ca2+ release. This, together with an impaired mitochondrial Ca2+ handling, results in insulin-mediated extra Ca2+ transients in ob/ob cardiomyocytes that may predispose for arrhythmias in vivo.
Obesity and type 2 diabetes are leading causes of coronary heart disease and heart failure (1eC4), and clinical and experimental studies have shown that diabetes is associated with altered cardiac function independent of vascular complications (5,6). Defective cellular Ca2+ handling is a fundamental problem in diabetes (7). For instance, diabetic cardiomyopathy is characterized by reduced levels of Ca2+-handling proteins and sarcoplasmic reticulum dysfunction leading to smaller and slower cytoplasmic Ca2+ transients (8).
Peripheral insulin resistance and hyperinsulinemia are hallmarks of type 2 diabetes and obesity. Insulin regulates various physiological processes in the heart including energy metabolism, contractility, protein expression, and ion transport (9). All insulin-mediated biological responses are consequences of the interaction between the insulin receptor, which belongs to the tyrosine kinase receptor family, and a complex array of downstream proteins (10,11). One central and early event in insulin signaling is the activation of phosphoinositide 3-kinase (PI3K), although insulin may also activate intracellular targets (e.g., MAP kinases) independent of PI3K activation. One tentative downstream target of PI3K is phospholipase C (PLC)- (12eC14). Activation of PLC- induces hydrolysis of phosphatidylinositol-bisphosphate (4,5) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (15). IP3 acts as a second messenger that mobilizes Ca2+ from intracellular stores via activation of specific IP3 receptors, whereas a major function of diacylglycerol is to activate protein kinase C (15). Insulin may also interfere with other modes of IP3 signaling in cardiomyocytes and, in this way, indirectly increase the IP3 concentration (16). Several studies have shown that IP3 can induce cardiac arrhythmias (17eC19), and hence alterations in IP3 signaling might be involved in diabetic cardiomyopathy. Elevated IP3 has also been linked to an increased Ca2+ influx into cells via a process named capacitive calcium entry (CCE) (20). Recently, CCE was shown to be important for the sustained elevation of cytoplasmic Ca2+ and hence, Ca2+-dependent cardiac remodelling after agonist stimulation of cultured neonatal rat ventricular myocytes (21). Furthermore, PLC-mediated CCE in cardiomyocytes was decreased by hyperglycemia-induced stimulation of the hexosamine pathway (22,23).
Diabetes is associated with mitochondrial dysfunction, increased production of reactive oxygen species (24), and decreased mitochondrial Ca2+ loading capacity (25,26). In the heart, altered mitochondrial Ca2+ uptake could have a deleterious effect on global Ca2+ homeostasis because mitochondria may act as a fixed spatial buffering system directly interacting with sarcoplasmic reticulum Ca2+ release (26).
The aim of the present study was to characterize insulin effects on Ca2+ homeostasis in normal mouse ventricular cardiomyocytes and to determine whether Ca2+ handling was changed in an animal model of type 2 diabetes, i.e., obese leptin-deficient ob/ob mice (27). We specifically focus on the role of IP3 and mitochondrial Ca2+ uptake. The results show marked differences between control and ob/ob cardiomyocytes that will increase the understanding of mechanisms underlying diabetic cardiomyopathy.
RESEARCH DESIGN AND METHODS
Human insulin was from Novo Nordisk. The membrane permeable acetoxymethyl ester forms of fluo-3 and rhod-2 were from Molecular Probes. 2-aminoethoxydiphenyl borate (2-APB), wortmannin, and laminin were from Sigma. A membrane-permeable acetoxymethyl form of IP3 (2,4,6-tri-O-butyryl-I[1,3,5]P3) was obtained from Calbiochem. This IP3 analog is more resistant to hydrolysis and metabolic degradation than the endogenous I(1,4,5)P3, but the specificity is uncertain since I(1,3,5)P3 itself has little affinity for the IP3 receptor, which indicates that the phosphate groups of the analog can migrate to other positions in the inositol molecule (28). All compounds were prepared as stock solutions in appropriate solvents. On the day of the experiment, stock solutions were diluted to the desired final concentration in the bath solution; when required, the same concentration of solvent was added to the control solution.
Animal model and cell isolation.
Young (3eC5 months) C57BL genetically obese male mice (ob/ob; body weight 49.7 ± 2.6 g) and their wild-type counterparts (body weight 27.0 ± 1.1 g) were housed at room temperature with free access to standard food pellets and water. Ob/ob mice are profoundly hyperinsulinemic and display moderate increases in serum glucose and lipids (27). To ascertain that our ob/ob mice were insulin resistant, we measured the insulin-mediated 2-deoxyglucose uptake in isolated extensor digitorum longus muscles (29) and found a significantly lower rate of uptake in ob/ob compared with wild-type muscles (90 ± 6 vs. 130 ± 5 eol · leC1 · mineC1, n = 8). One mouse was killed in the morning (9:00 A.M.) of each experimental day by rapid neck disarticulation, and the heart was excised. Single cardiomyocytes were isolated from the ventricles following the protocols developed by the Alliance for Cellular Signaling (Procedure Protocol ID PP00000 125) (30). All experiments were approved by the Stockholm North local ethical committee.
Measurement of cytosolic Ca2+.
The free cytosolic [Ca2+] was measured with the fluorescent Ca2+ indicator fluo-3. Isolated cardiomyocytes were incubated in Dulbecco’s modified Eagle's medium (Sigma) containing 20 eol/l fluo-3 AM for 40 min at room temperature followed by 10 min in medium without fluo-3. After being loaded, cardiomyocytes were plated on laminin-coated glass coverslips that made up the bottom of the perfusion chamber. Cells were superfused with standard Tyrode solution (mmol/l): 121 NaCl, 5.0 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.4 NaH2PO4, 24 NaHCO3, 0.1 EDTA, and 5.5 glucose. The solution was bubbled with 5% CO2/95% O2, which gives a bath pH of 7.4. Experiments were performed at room temperature (24°C). Cells were stimulated with 1- to 2-ms current pulses delivered via two platinum electrodes (one on each side of the perfusion chamber). Changes in fluo-3 fluorescence were measured with confocal microscopy using a BioRad MRC 1024 unit (BioRad Microscopy Division, Hertfordshire, U.K.) attached to a Nikon Diaphot 200 inverted microscope with a Nikon Plan Apo 40x oil immersion objective (numerical aperture 1.3). Experiments were performed in the line-scan mode (6-ms intervals), and scanning was performed along the long axis of the cell. Excitation was at 488 nm, and the emitted light was collected through a 522-nm narrow band filter. The laser power used (3eC6% of the maximum) did not have any noticeable deleterious effect on the fluorescent signal or cell function over the time course of an experiment. To enable comparisons between cells, changes in the fluorescence signal (F) were divided by the fluorescence immediately before the stimulation pulse at 1-Hz stimulation (F0). The time course of Ca2+ transients was assessed by measuring the time to peak (TTP); the half-width (D1/2), i.e., the duration at 50% of F; and the time constant () of the exponential part of the decay phase, ignoring the initial decline that clearly diverged from a mono-exponential function (e.g., see wild-type transient in Fig. 1C).
Measurements of mitochondrial Ca2+.
Rhod-2 was used to measure mitochondrial Ca2+ (31). Isolated cardiomyocytes were loaded with rhod-2 AM (10 eol/l) for 30 min at 4°C followed by washout and at least 1 h rest at room temperature. During the last 30 min of this rest period, cells were also loaded with fluo-3 AM (10 eol/l) for 20 min followed by 10-min washout. Cells were continuously stimulated at 1 Hz, ensuring stable mitochondrial Ca2+ transients (31). Fluo-3 and rhod-2 fluorescence signals were obtained in the same line scan by excitation at 488 and 568 nm and measuring the emitted light at 522 and 585 nm, respectively. The fluo-3 signal was then used to subtract the cytosolic component of the rhod-2 signal leaving only the mitochondrial component. Control experiments were performed on wild-type cardiomyocytes where cells were only loaded with rhod-2 AM for 1 h at 4°C followed by at least 1 h washout at room temperature, a procedure that has been found to optimize mitochondrial rhod-2 loading in cardiomyocytes (31). These two procedures to assess mitochondrial Ca2+ gave very similar results that differed significantly (P < 0.05) from the cytosolic fluo-3 signals: under control conditions, the amplitude (F/F0) was 3.1 ± 0.9 for the cytosolic fluo-3 signal vs. 1.8 ± 0.5 (n = 12) and 1.9 ± 0.6 (n = 8) for the mitochondrial fluo-3-subtracted and rhod-2 only signals, respectively; the time to peak was 25 ± 3 ms with fluo-3 vs. 115 ± 21 and 121 ± 20 ms in the two sets of rhod-2 measurements.
Measurements of IP3.
Cells were incubated in medium containing 10 mmol/l LiCl2 in the absence or presence of insulin (60 nmol/l) for 15 min at room temperature. During the last 5 min, cells were allowed to settle at the bottom of the incubation tubes. The medium was removed and ice-cold 0.5 mol/l perchloric acid was added to the cells. The mixture was vortexed and kept in an ice slurry for 20 min. Thereafter, the acid extract was centrifuged (10,000g at 4°C for 15 min). The pellet was extracted with 1 mol/l NaOH for subsequent analysis of protein (Bio-Rad method). The supernatant was neutralized with ice-cold 2.2 mol/l KHCO3 and centrifuged again. The final supernatant was analyzed for IP3 using the [3H] Biotrak Assay System (Amersham Biosciences, Piscataway, NJ).
Immunoprecipitation and Western blot analyses.
Frozen hearts were thawed and left ventricles homogenized in lysis buffer comprising 20 mmol/l HEPES, pH 7.6, 150 mmol/l NaCl, 20% glycerol (vol/vol), 5 mmol/l EDTA, 1 mmol/l Na3V04, 25 mmol/l KF, 0.5% Triton X-100 (vol/vol), and protease inhibitor cocktail (Roche). Lysates were cleared by centrifugation at 10,000g for 10 min at 4°C. The protein content was determined using the Bradford method (BioRad). Equal amounts of protein were incubated with primary antibodies for 5 min at room temperature, followed by addition of 30 e蘬 of protein G agarose suspension (Santa Cruz Biotechnology, Santa Cruz, CA) for at least 4 h at 4°C with rotation. Primary antibodies used were anti-IP3 receptor type 1 (anti-IP3R1; gift from K. Rietdorf, L. Roderick, and M. Bootman at the Babraham Institute, Cambridge, U.K.) and anti-IP3 receptor type 2 (anti-IP3R2; Santa Cruz). After washing three times with lysis buffer, samples were heated with SDS-PAGE sample buffer for 10 min at 70°C and proteins separated by 3eC8% Tris-acetate gradient gels (Invitrogen) and transferred onto a polyvinylidine fluoride membrane (BioRad). Membranes were blocked in 5% (wt/vol) nonfat milk in Tris-buffered saline containing 0.05% Tween 20 followed by incubation with primary antibodies (anti-IP3R1, 1:6,000 dilution; anti-IP3R2, 1:100 dilution). Blots were then incubated with secondary horseradish peroxidaseeCconjugated antibody (anti-rabbit Ig, 1:40,000 [Amersham]; anti-goat Ig, 1:5,000 [BioRad]), and immunoreactive bands were visualized using enhanced chemiluminescence (SuperSignal; Pierce Biotechnology, Rockford, IL).
Statistics.
Stored confocal images were analyzed with ImageJ (National Institutes of Health [available at http://rsb.info.nih.gov/ij/]). Data are presented as mean ± SE. Statistics were performed using Student’s t test (for paired or unpaired samples) and one-way ANOVA when three or more groups were compared, along with a Newman-Keuls post hoc test. Differences were considered significant when the P value was <0.05.
RESULTS
Effects of insulin on cytoplasmic Ca2+ transients.
We investigated characteristics of Ca2+ transients recorded during 1-Hz stimulation in wild-type and ob/ob mouse ventricular cardiomyocytes. Under control conditions, the amplitude of Ca2+ transients was smaller and the decay phase was slower in ob/ob compared with wild-type cardiomyocytes (Fig. 1). Contractions were also markedly weaker in ob/ob cells that shortened by 7.1 ± 0.8% of their resting length (n = 21) compared with 15.5 ± 1.8% (n = 33) in wild-type cells. Application of insulin (60 nmol/l = 10 mU/ml) significantly increased the Ca2+ transient amplitude by 30% in wild-type cells, whereas there was no significant effect on the time course (Table 1). In ob/ob cardiomyocytes, on the other hand, insulin did not produce any significant change of the Ca2+ transient amplitude, but the transient became broader. This broadening of the transient was due to an increased time to peak and slowed onset of the decay phase, whereas the rate of decline during the final phase, if anything, tended to be faster in the presence of insulin (Table 1). Application of insulin triggered frequent extra Ca2+ transients during the final part of the decay phase in 10 of 18 ob/ob cardiomyocytes (Fig. 2 ). Such extra Ca2+ transients were not observed under control conditions in ob/ob cells, nor were they seen in wild-type cells even in the presence of insulin. Inhibition of PI3K with wortmannin (0.5 eol/l) completely blocked the effects of insulin on Ca2+ transients in both wild-type and ob/ob cells (Table 1), which suggests that the effects of insulin occurred via the PI3K-dependent signaling pathway (10,11).
The concentration of insulin used in the experiments described above (60 nmol/l) is higher than that observed in vivo. We therefore also tested the effects of 6 nmol/l insulin, which is in the same range as the serum concentration of fed ob/ob mice (1.6 nmol/l) (27). The effects of 6 nmol/l insulin on Ca2+ transients were qualitatively the same as those observed with 60 nmol/l in both wild-type and ob/ob cells. Thus, in wild-type cardiomyocytes (n = 5), the peak amplitude of the Ca2+ transient increased from 3.5 ±. 0.2 in control to 4.1 ± 0.2 with 6 nmol/l insulin (P < 0.05), whereas the time to peak, half width, and decay time constant of the transient were not affected. In ob/ob cells (n = 5), application of 6 nmol/l insulin increased (P < 0.05) the time to peak (21.8 ± 2.9 ms in control vs. 42.0 ± 6.4 ms with insulin) and the half width (194 ± 12 vs. 209 ± 7 ms), whereas the peak amplitude and the decay time constant were not altered.
Effects of IP3 on cytoplasmic Ca2+ transients.
We also tested the effect of a membrane-permeant IP3 analog (10 eol/l) on Ca2+ transients. Application of the IP3 analog had no significant effect on the amplitude of Ca2+ transients in ob/ob or wild-type cardiomyocytes (Table 1). However, it produced broader Ca2+ transients in both groups due to an increased time to peak and slowed early decay phase, whereas the rate of the final decay was not affected. Moreover, the IP3 analog induced extra Ca2+ transients in 8 of 13 ob/ob cells, whereas only 2 of 18 wild-type cells showed such transients (Figs. 2B and D).
To further investigate the possible role of IP3 in insulin signaling, cells were preincubated for 15 min with 2-APB (30 eol/l), a frequently used inhibitor of IP3 receptors. 2-APB prevented the insulin-mediated slowing of the Ca2+ transient in ob/ob cells; for instance, in the presence of 2-APB, the time to peak was 28.6 ± 4.0 ms (n = 15) without and 33.2 ± 6.0 ms (n = 8) with insulin (P > 0.05, unpaired t test). Furthermore, no extra Ca2+ transients were triggered by insulin in the presence of 2-APB (Fig. 3A). 2-APB also prevented the IP3-mediated slowing of the Ca2+ transient (time to peak 34.3 ± 7.4 ms [n = 7]) and prevented the induction of extra Ca2+ transients in ob/ob cells (Fig. 3B). However, 2-APB did not inhibit the insulin-induced increase in the Ca2+ transient amplitude in wild-type cells: F/F0 was 3.1 ± 0.5 without and 4.0 ± 0.3 with insulin, respectively (n = 7).
Expression of IP3 receptors and insulin effects on the IP3 concentration.
We used immunoprecipitation and Western blots to measure the expression of IP3 receptor type 1 and type 2 in ventricles from wild-type and ob/ob mice, of which type 2 is the predominant isoform in cardiomyocytes (32). The mean expression of IP3 receptor type 2 was not different between ob/ob and wild-type ventricles (P = 0.41) (Fig. 3C). Moreover, there was no difference in the expression of IP3 receptor type 1 (data not shown).
The IP3 concentration was measured in wild-type and ob/ob cardiomyocytes in the absence and presence of insulin. Insulin had no effect on the IP3 concentration in wild-type cells, whereas it significantly (P < 0.05) increased the concentration by 30% in ob/ob cells (Fig. 3D).
Spontaneous Ca2+ waves.
Since IP3 has been shown to generate arrhythmias and spontaneous Ca2+ events in cardiomyocytes (17,19), we studied the occurrence of spontaneous propagating Ca2+ waves in resting (not paced) cardiomyocytes. Few spontaneous Ca2+ waves were observed under control conditions. Application of insulin (60 nmol/l) significantly increased the frequency of waves in both wild-type and ob/ob cells (Fig. 4A). The insulin-induced increase in the frequency of spontaneous waves was fully reversed after 15-min washout in both cell groups (data not shown). Pretreatment with wortmannin or 2-APB completely blocked the insulin-mediated increase in wave frequency (Fig. 4B).
The effect of IP3 was also tested under the same experimental conditions. IP3 application resulted in frequent spontaneous propagating Ca2+ waves, and the effect was completely blocked by 2-APB (Fig. 4B).
Mitochondrial Ca2+ transient.
Insulin had markedly different effects on cytosolic Ca2+ transients in wild-type and ob/ob cardiomyocytes, producing an increase in the amplitude in wild-type cells, whereas Ca2+ transients were broadened and extra transients occured during relaxation in ob/ob cells (see Table 1 and Fig. 2). Altered mitochondrial Ca2+ uptake in a beat-to-beat manner may have a role in this difference between wild-type and ob/ob cardiomyocytes, since mitochondria are known to contribute to the shaping of Ca2+ signals in cardiomyocytes (33) and diabetes is associated with impaired mitochondrial function (27). We therefore recorded transients of mitochondrial rhod-2 fluorescence at 1-Hz stimulation in the absence or presence of insulin (60 nmol/l). There was no difference between wild-type and ob/ob cells regarding the amplitude of mitochondrial Ca2+ transients under control conditions. However, the transients were significantly (P < 0.05) slower in ob/ob compared with wild-type cells (Fig. 5, top), with the time to peak and half-width being 182 ± 19 and 325 ± 24 ms in ob/ob cells (n = 14) vs. 115 ± 21 and 222 ± 20 ms in wild-type cells (n = 12), respectively. Application of insulin significantly increased the amplitude of mitochondrial Ca2+ transients in wild-type cells, whereas the amplitude was not changed in ob/ob cells (Fig. 5, bottom). Application of the IP3 analog also increased the amplitude in wild-type but not in ob/ob cells. Thus, the dynamic mitochondrial Ca2+ buffering during insulin or IP3 exposure was blunted in ob/ob cardiomyocytes.
DISCUSSION
Defects in the intracellular Ca2+ handling appear to be a generalized problem in diabetes (7). In the present study, we compared critical aspects of intracellular Ca2+ handling in ventricular cardiomyocytes of wild-type and ob/ob mice, a model of obesity and type 2 diabetes. The major novel results are 1) insulin triggers extra Ca2+ transients in ob/ob but not in wild-type cardiomyocytes, which indicates an increased susceptibility for developing arrhythmias in ob/ob hearts; 2) the effects of insulin on Ca2+ transients are reproduced by application of a membrane-permeant IP3 analog in ob/ob but not in wild-type cardiomyocytes; 3) in ob/ob cardiomyocytes, the effects of insulin and IP3 were blocked by 2-APB, and insulin increased the IP3 concentration; and 4) mitochondrial Ca2+ handling was impaired in ob/ob cardiomyocytes.
Ca2+ transients in obesity and type 2 diabetes.
Under control conditions, electrically evoked Ca2+ transients were smaller and slower in ob/ob cells, which may account for the decrease in peak contraction and slowed relaxation observed in different models of obesity and type 2 diabetes (34eC36). Moreover, a recent study on cardiomyocytes isolated from obese, type 2 diabetic mice lacking functional leptin receptors (db/db mice) also showed significantly smaller and slower Ca2+ transients in comparison with control cells (37).
Ca2+ transients in cardiomyocytes are mediated by Ca2+ influx through voltage-activated L-type Ca2+ channels, which activate sarcoplasmic reticulum Ca2+ release channels (ryanodine receptor-2) via a process known as Ca2+-induced Ca2+ release (38,39). Relaxation occurs when Ca2+ release is stopped and Ca2+ removed from the cytoplasm. This occurs predominantly by active reuptake into the sarcoplasmic reticulum by Ca2+ ATPase 2A, but Ca2+ extrusion out of the cell via Na+/Ca2+ exchange also contributes (39). The Ca2+ transient amplitude increased when insulin was applied in wild-type but not in ob/ob cardiomyocytes. This difference might reflect an inability of insulin to increase the L-type Ca2+ current in type 2 diabetes, whereas the inotropic effect of insulin is at least partly attributable to an increased L-type Ca2+ current in normal subjects (40eC42). This suggestion fits with our finding that 2-APB, which preferentially inhibits IP3-mediated signaling (43), had no effect on the insulin-induced increase in Ca2+ transient amplitude in wild-type cells, whereas it blocked the effects of insulin on Ca2+ handling in ob/ob cells. Thus, the insulin-induced increase of the Ca2+ transient amplitude in wild-type cells appears not to be mediated via IP3.
Possible role of IP3 in the insulin signaling.
Insulin application resulted in slowed Ca2+ transient kinetics and the appearance of frequent extra Ca2+ transients in ob/ob but not in wild-type cardiomyocytes. The insulin-mediated slowing of Ca2+ transients in ob/ob cells was due to an increased time to peak and slowed onset of the decay phase, whereas the rate of decline during the exponential decay phase was, if anything, increased (see Table 1). This indicates that insulin prolonged the sarcoplasmic reticulum Ca2+ release process whereas it had little effect on Ca2+ removal, which is dominated by the active sarcoplasmic reticulum Ca2+ reuptake (39). Application of a membrane-permeant IP3 analog gave results qualitatively the same as those of insulin in ob/ob cells; that is, there was no significant effect on the Ca2+ transient amplitude or the exponential decay rate, whereas the time to peak was increased, the early decay phase was slowed, and frequent extra Ca2+ transients were produced. Thus, both insulin and IP3 apparently increased the duration of action potential-mediated sarcoplasmic reticulum Ca2+ release in ob/ob cells, and this was accompanied by the triggering of extra Ca2+ transients. In accordance with these findings, insulin application caused an 30% increase in the IP3 concentration in ob/ob cardiomyocytes. On the other hand, the expression of type 1 and 2 IP3 receptors was not different between ob/ob and wild-type ventricles, and hence this cannot explain the differences between the two groups regarding the response to insulin and IP3.
The involvement of IP3 in insulin signaling in cardiomyocytes is further supported by the fact that the insulin- and IP3-mediated effects on electrically evoked Ca2+ transients in ob/ob cells were prevented by preincubation with 2-APB (see Fig. 3), one important action of which is to inhibit IP3 receptors (43). Furthermore, both insulin and IP3 induced spontaneous Ca2+ waves in rested wild-type and ob/ob cardiomyocytes, and this effect was fully blocked by 2-APB (Fig. 4). Finally, several studies have shown that IP3 can induce cardiac arrhythmias (17,18,19), which fits with the occurrence of insulin- and IP3-induced extra Ca2+ transients in ob/ob cells.
Possible role of defective mitochondrial function in impaired intracellular Ca2+ handling.
An increase in mitochondrial Ca2+ may stimulate oxidative metabolism via activation of enzymes involved in mitochondrial energy production (44eC46). Dynamic changes in mitochondrial Ca2+ are driven by the cytosolic Ca2+ transients in beating cardiomyocytes (31), thus providing a simple and elegant link between work and energy supply. Furthermore, a marked increase in mitochondrial Ca2+ in response to IP3-linked stimuli has been observed in a large variety of cell types (47). In terms of regulating global cytosolic Ca2+ handling, mitochondria are believed to act as a spatial buffering system that can blunt or slow propagating Ca2+ waves (48,49), as well as directly controlling Ca2+ release via IP3 receptors (49). Conversely, impaired mitochondrial Ca2+ accumulation may have deleterious effects by increasing cytosolic Ca2+ (49). In the present study, we showed slowed mitochondrial Ca2+ uptake in ob/ob cardiomyocytes compared with wild-type cells. Furthermore, the mitochondrial Ca2+ uptake did not increase in response to insulin or IP3 in ob/ob cells. Thus, the impaired mitochondrial Ca2+ uptake in ob/ob cells may contribute to the larger slowing of Ca2+ transients induced by insulin and IP3 in these cells, as well as the occurrence of extra Ca2+ transients.
We used rhod-2 to monitor mitochondrial Ca2+, and although this nonratiometric dye can readily measure transient changes in Ca2+, it is less suitable for detecting changes in basal mitochondrial Ca2+ accumulation. Thus, we are not able to distinguish between a basal mitochondrial Ca2+ overload or primary changes in mitochondrial Ca2+ flux kinetics as the major mechanism underlying the alterations observed in ob/ob cardiomyocytes.
Based on the present results, we propose the following model to explain the impaired Ca2+ handling in ob/ob cardiomyocytes: insulin increases the IP3 concentration in ob/ob cardiomyocytes, which prolongs electrically evoked sarcoplasmic reticulum Ca2+ release. Mitochondrial Ca2+ uptake is impaired in ob/ob cardiomyocytes, which decreases the ability to buffer the extra Ca2+ released during physiological challenges. Together, these defects in ob/ob cardiomyocytes cause a slowing of the Ca2+ transient and increase the probability of extra Ca2+ transients that may predispose for arrhythmias in vivo.
ACKNOWLEDGMENTS
This study was supported by the Swedish Research Council (project numbers 10842 and 14453), the Swedish Heart and Lung Foundation, Biovitrum Partner Fund, the Swedish Diabetes Foundation, and Funds at the Karolinska Institutet.
FOOTNOTES
A.K. and H.W. have received grant support from Biovitrum Partner.
Key Words: 2-APB, 2-aminoethoxydiphenyl borate CCE, capacitive calcium entry IP3, inositol 1,4,5-trisphosphate PI3K, phosphoinositide 3-kinase PLC, phospholipase C
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ABSTRACT
Obesity, insulin resistance, and type 2 diabetes are leading causes of heart failure, and defective cellular Ca2+ handling seems to be a fundamental problem in diabetes. Therefore, we studied the effect of insulin on Ca2+ homeostasis in normal, freshly isolated mouse ventricular cardiomyocytes and whether Ca2+ handling was changed in an animal model of obesity and type 2 diabetes, ob/ob mice. Electrically evoked Ca2+ transients were smaller and slower in ob/ob compared with wild-type cardiomyocytes. Application of insulin (6 or 60 nmol/l) increased the amplitude of Ca2+ transients in wild-type cells by 30%, whereas it broadened the transients and triggered extra Ca2+ transients in ob/ob cells. The effects of insulin in ob/ob cells could be reproduced by application of a membrane-permeant inositol trisphosphate (IP3) analog and blocked by a frequently used IP3 receptor inhibitor, 2-aminoethoxydiphenyl borate. In ob/ob cardiomyocytes, insulin increased the IP3 concentration and mitochondrial Ca2+ handling was impaired. In conclusion, we propose a model where insulin increases IP3 in ob/ob cardiomyocytes, which prolongs the electrically evoked Ca2+ release. This, together with an impaired mitochondrial Ca2+ handling, results in insulin-mediated extra Ca2+ transients in ob/ob cardiomyocytes that may predispose for arrhythmias in vivo.
Obesity and type 2 diabetes are leading causes of coronary heart disease and heart failure (1eC4), and clinical and experimental studies have shown that diabetes is associated with altered cardiac function independent of vascular complications (5,6). Defective cellular Ca2+ handling is a fundamental problem in diabetes (7). For instance, diabetic cardiomyopathy is characterized by reduced levels of Ca2+-handling proteins and sarcoplasmic reticulum dysfunction leading to smaller and slower cytoplasmic Ca2+ transients (8).
Peripheral insulin resistance and hyperinsulinemia are hallmarks of type 2 diabetes and obesity. Insulin regulates various physiological processes in the heart including energy metabolism, contractility, protein expression, and ion transport (9). All insulin-mediated biological responses are consequences of the interaction between the insulin receptor, which belongs to the tyrosine kinase receptor family, and a complex array of downstream proteins (10,11). One central and early event in insulin signaling is the activation of phosphoinositide 3-kinase (PI3K), although insulin may also activate intracellular targets (e.g., MAP kinases) independent of PI3K activation. One tentative downstream target of PI3K is phospholipase C (PLC)- (12eC14). Activation of PLC- induces hydrolysis of phosphatidylinositol-bisphosphate (4,5) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (15). IP3 acts as a second messenger that mobilizes Ca2+ from intracellular stores via activation of specific IP3 receptors, whereas a major function of diacylglycerol is to activate protein kinase C (15). Insulin may also interfere with other modes of IP3 signaling in cardiomyocytes and, in this way, indirectly increase the IP3 concentration (16). Several studies have shown that IP3 can induce cardiac arrhythmias (17eC19), and hence alterations in IP3 signaling might be involved in diabetic cardiomyopathy. Elevated IP3 has also been linked to an increased Ca2+ influx into cells via a process named capacitive calcium entry (CCE) (20). Recently, CCE was shown to be important for the sustained elevation of cytoplasmic Ca2+ and hence, Ca2+-dependent cardiac remodelling after agonist stimulation of cultured neonatal rat ventricular myocytes (21). Furthermore, PLC-mediated CCE in cardiomyocytes was decreased by hyperglycemia-induced stimulation of the hexosamine pathway (22,23).
Diabetes is associated with mitochondrial dysfunction, increased production of reactive oxygen species (24), and decreased mitochondrial Ca2+ loading capacity (25,26). In the heart, altered mitochondrial Ca2+ uptake could have a deleterious effect on global Ca2+ homeostasis because mitochondria may act as a fixed spatial buffering system directly interacting with sarcoplasmic reticulum Ca2+ release (26).
The aim of the present study was to characterize insulin effects on Ca2+ homeostasis in normal mouse ventricular cardiomyocytes and to determine whether Ca2+ handling was changed in an animal model of type 2 diabetes, i.e., obese leptin-deficient ob/ob mice (27). We specifically focus on the role of IP3 and mitochondrial Ca2+ uptake. The results show marked differences between control and ob/ob cardiomyocytes that will increase the understanding of mechanisms underlying diabetic cardiomyopathy.
RESEARCH DESIGN AND METHODS
Human insulin was from Novo Nordisk. The membrane permeable acetoxymethyl ester forms of fluo-3 and rhod-2 were from Molecular Probes. 2-aminoethoxydiphenyl borate (2-APB), wortmannin, and laminin were from Sigma. A membrane-permeable acetoxymethyl form of IP3 (2,4,6-tri-O-butyryl-I[1,3,5]P3) was obtained from Calbiochem. This IP3 analog is more resistant to hydrolysis and metabolic degradation than the endogenous I(1,4,5)P3, but the specificity is uncertain since I(1,3,5)P3 itself has little affinity for the IP3 receptor, which indicates that the phosphate groups of the analog can migrate to other positions in the inositol molecule (28). All compounds were prepared as stock solutions in appropriate solvents. On the day of the experiment, stock solutions were diluted to the desired final concentration in the bath solution; when required, the same concentration of solvent was added to the control solution.
Animal model and cell isolation.
Young (3eC5 months) C57BL genetically obese male mice (ob/ob; body weight 49.7 ± 2.6 g) and their wild-type counterparts (body weight 27.0 ± 1.1 g) were housed at room temperature with free access to standard food pellets and water. Ob/ob mice are profoundly hyperinsulinemic and display moderate increases in serum glucose and lipids (27). To ascertain that our ob/ob mice were insulin resistant, we measured the insulin-mediated 2-deoxyglucose uptake in isolated extensor digitorum longus muscles (29) and found a significantly lower rate of uptake in ob/ob compared with wild-type muscles (90 ± 6 vs. 130 ± 5 eol · leC1 · mineC1, n = 8). One mouse was killed in the morning (9:00 A.M.) of each experimental day by rapid neck disarticulation, and the heart was excised. Single cardiomyocytes were isolated from the ventricles following the protocols developed by the Alliance for Cellular Signaling (Procedure Protocol ID PP00000 125) (30). All experiments were approved by the Stockholm North local ethical committee.
Measurement of cytosolic Ca2+.
The free cytosolic [Ca2+] was measured with the fluorescent Ca2+ indicator fluo-3. Isolated cardiomyocytes were incubated in Dulbecco’s modified Eagle's medium (Sigma) containing 20 eol/l fluo-3 AM for 40 min at room temperature followed by 10 min in medium without fluo-3. After being loaded, cardiomyocytes were plated on laminin-coated glass coverslips that made up the bottom of the perfusion chamber. Cells were superfused with standard Tyrode solution (mmol/l): 121 NaCl, 5.0 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.4 NaH2PO4, 24 NaHCO3, 0.1 EDTA, and 5.5 glucose. The solution was bubbled with 5% CO2/95% O2, which gives a bath pH of 7.4. Experiments were performed at room temperature (24°C). Cells were stimulated with 1- to 2-ms current pulses delivered via two platinum electrodes (one on each side of the perfusion chamber). Changes in fluo-3 fluorescence were measured with confocal microscopy using a BioRad MRC 1024 unit (BioRad Microscopy Division, Hertfordshire, U.K.) attached to a Nikon Diaphot 200 inverted microscope with a Nikon Plan Apo 40x oil immersion objective (numerical aperture 1.3). Experiments were performed in the line-scan mode (6-ms intervals), and scanning was performed along the long axis of the cell. Excitation was at 488 nm, and the emitted light was collected through a 522-nm narrow band filter. The laser power used (3eC6% of the maximum) did not have any noticeable deleterious effect on the fluorescent signal or cell function over the time course of an experiment. To enable comparisons between cells, changes in the fluorescence signal (F) were divided by the fluorescence immediately before the stimulation pulse at 1-Hz stimulation (F0). The time course of Ca2+ transients was assessed by measuring the time to peak (TTP); the half-width (D1/2), i.e., the duration at 50% of F; and the time constant () of the exponential part of the decay phase, ignoring the initial decline that clearly diverged from a mono-exponential function (e.g., see wild-type transient in Fig. 1C).
Measurements of mitochondrial Ca2+.
Rhod-2 was used to measure mitochondrial Ca2+ (31). Isolated cardiomyocytes were loaded with rhod-2 AM (10 eol/l) for 30 min at 4°C followed by washout and at least 1 h rest at room temperature. During the last 30 min of this rest period, cells were also loaded with fluo-3 AM (10 eol/l) for 20 min followed by 10-min washout. Cells were continuously stimulated at 1 Hz, ensuring stable mitochondrial Ca2+ transients (31). Fluo-3 and rhod-2 fluorescence signals were obtained in the same line scan by excitation at 488 and 568 nm and measuring the emitted light at 522 and 585 nm, respectively. The fluo-3 signal was then used to subtract the cytosolic component of the rhod-2 signal leaving only the mitochondrial component. Control experiments were performed on wild-type cardiomyocytes where cells were only loaded with rhod-2 AM for 1 h at 4°C followed by at least 1 h washout at room temperature, a procedure that has been found to optimize mitochondrial rhod-2 loading in cardiomyocytes (31). These two procedures to assess mitochondrial Ca2+ gave very similar results that differed significantly (P < 0.05) from the cytosolic fluo-3 signals: under control conditions, the amplitude (F/F0) was 3.1 ± 0.9 for the cytosolic fluo-3 signal vs. 1.8 ± 0.5 (n = 12) and 1.9 ± 0.6 (n = 8) for the mitochondrial fluo-3-subtracted and rhod-2 only signals, respectively; the time to peak was 25 ± 3 ms with fluo-3 vs. 115 ± 21 and 121 ± 20 ms in the two sets of rhod-2 measurements.
Measurements of IP3.
Cells were incubated in medium containing 10 mmol/l LiCl2 in the absence or presence of insulin (60 nmol/l) for 15 min at room temperature. During the last 5 min, cells were allowed to settle at the bottom of the incubation tubes. The medium was removed and ice-cold 0.5 mol/l perchloric acid was added to the cells. The mixture was vortexed and kept in an ice slurry for 20 min. Thereafter, the acid extract was centrifuged (10,000g at 4°C for 15 min). The pellet was extracted with 1 mol/l NaOH for subsequent analysis of protein (Bio-Rad method). The supernatant was neutralized with ice-cold 2.2 mol/l KHCO3 and centrifuged again. The final supernatant was analyzed for IP3 using the [3H] Biotrak Assay System (Amersham Biosciences, Piscataway, NJ).
Immunoprecipitation and Western blot analyses.
Frozen hearts were thawed and left ventricles homogenized in lysis buffer comprising 20 mmol/l HEPES, pH 7.6, 150 mmol/l NaCl, 20% glycerol (vol/vol), 5 mmol/l EDTA, 1 mmol/l Na3V04, 25 mmol/l KF, 0.5% Triton X-100 (vol/vol), and protease inhibitor cocktail (Roche). Lysates were cleared by centrifugation at 10,000g for 10 min at 4°C. The protein content was determined using the Bradford method (BioRad). Equal amounts of protein were incubated with primary antibodies for 5 min at room temperature, followed by addition of 30 e蘬 of protein G agarose suspension (Santa Cruz Biotechnology, Santa Cruz, CA) for at least 4 h at 4°C with rotation. Primary antibodies used were anti-IP3 receptor type 1 (anti-IP3R1; gift from K. Rietdorf, L. Roderick, and M. Bootman at the Babraham Institute, Cambridge, U.K.) and anti-IP3 receptor type 2 (anti-IP3R2; Santa Cruz). After washing three times with lysis buffer, samples were heated with SDS-PAGE sample buffer for 10 min at 70°C and proteins separated by 3eC8% Tris-acetate gradient gels (Invitrogen) and transferred onto a polyvinylidine fluoride membrane (BioRad). Membranes were blocked in 5% (wt/vol) nonfat milk in Tris-buffered saline containing 0.05% Tween 20 followed by incubation with primary antibodies (anti-IP3R1, 1:6,000 dilution; anti-IP3R2, 1:100 dilution). Blots were then incubated with secondary horseradish peroxidaseeCconjugated antibody (anti-rabbit Ig, 1:40,000 [Amersham]; anti-goat Ig, 1:5,000 [BioRad]), and immunoreactive bands were visualized using enhanced chemiluminescence (SuperSignal; Pierce Biotechnology, Rockford, IL).
Statistics.
Stored confocal images were analyzed with ImageJ (National Institutes of Health [available at http://rsb.info.nih.gov/ij/]). Data are presented as mean ± SE. Statistics were performed using Student’s t test (for paired or unpaired samples) and one-way ANOVA when three or more groups were compared, along with a Newman-Keuls post hoc test. Differences were considered significant when the P value was <0.05.
RESULTS
Effects of insulin on cytoplasmic Ca2+ transients.
We investigated characteristics of Ca2+ transients recorded during 1-Hz stimulation in wild-type and ob/ob mouse ventricular cardiomyocytes. Under control conditions, the amplitude of Ca2+ transients was smaller and the decay phase was slower in ob/ob compared with wild-type cardiomyocytes (Fig. 1). Contractions were also markedly weaker in ob/ob cells that shortened by 7.1 ± 0.8% of their resting length (n = 21) compared with 15.5 ± 1.8% (n = 33) in wild-type cells. Application of insulin (60 nmol/l = 10 mU/ml) significantly increased the Ca2+ transient amplitude by 30% in wild-type cells, whereas there was no significant effect on the time course (Table 1). In ob/ob cardiomyocytes, on the other hand, insulin did not produce any significant change of the Ca2+ transient amplitude, but the transient became broader. This broadening of the transient was due to an increased time to peak and slowed onset of the decay phase, whereas the rate of decline during the final phase, if anything, tended to be faster in the presence of insulin (Table 1). Application of insulin triggered frequent extra Ca2+ transients during the final part of the decay phase in 10 of 18 ob/ob cardiomyocytes (Fig. 2 ). Such extra Ca2+ transients were not observed under control conditions in ob/ob cells, nor were they seen in wild-type cells even in the presence of insulin. Inhibition of PI3K with wortmannin (0.5 eol/l) completely blocked the effects of insulin on Ca2+ transients in both wild-type and ob/ob cells (Table 1), which suggests that the effects of insulin occurred via the PI3K-dependent signaling pathway (10,11).
The concentration of insulin used in the experiments described above (60 nmol/l) is higher than that observed in vivo. We therefore also tested the effects of 6 nmol/l insulin, which is in the same range as the serum concentration of fed ob/ob mice (1.6 nmol/l) (27). The effects of 6 nmol/l insulin on Ca2+ transients were qualitatively the same as those observed with 60 nmol/l in both wild-type and ob/ob cells. Thus, in wild-type cardiomyocytes (n = 5), the peak amplitude of the Ca2+ transient increased from 3.5 ±. 0.2 in control to 4.1 ± 0.2 with 6 nmol/l insulin (P < 0.05), whereas the time to peak, half width, and decay time constant of the transient were not affected. In ob/ob cells (n = 5), application of 6 nmol/l insulin increased (P < 0.05) the time to peak (21.8 ± 2.9 ms in control vs. 42.0 ± 6.4 ms with insulin) and the half width (194 ± 12 vs. 209 ± 7 ms), whereas the peak amplitude and the decay time constant were not altered.
Effects of IP3 on cytoplasmic Ca2+ transients.
We also tested the effect of a membrane-permeant IP3 analog (10 eol/l) on Ca2+ transients. Application of the IP3 analog had no significant effect on the amplitude of Ca2+ transients in ob/ob or wild-type cardiomyocytes (Table 1). However, it produced broader Ca2+ transients in both groups due to an increased time to peak and slowed early decay phase, whereas the rate of the final decay was not affected. Moreover, the IP3 analog induced extra Ca2+ transients in 8 of 13 ob/ob cells, whereas only 2 of 18 wild-type cells showed such transients (Figs. 2B and D).
To further investigate the possible role of IP3 in insulin signaling, cells were preincubated for 15 min with 2-APB (30 eol/l), a frequently used inhibitor of IP3 receptors. 2-APB prevented the insulin-mediated slowing of the Ca2+ transient in ob/ob cells; for instance, in the presence of 2-APB, the time to peak was 28.6 ± 4.0 ms (n = 15) without and 33.2 ± 6.0 ms (n = 8) with insulin (P > 0.05, unpaired t test). Furthermore, no extra Ca2+ transients were triggered by insulin in the presence of 2-APB (Fig. 3A). 2-APB also prevented the IP3-mediated slowing of the Ca2+ transient (time to peak 34.3 ± 7.4 ms [n = 7]) and prevented the induction of extra Ca2+ transients in ob/ob cells (Fig. 3B). However, 2-APB did not inhibit the insulin-induced increase in the Ca2+ transient amplitude in wild-type cells: F/F0 was 3.1 ± 0.5 without and 4.0 ± 0.3 with insulin, respectively (n = 7).
Expression of IP3 receptors and insulin effects on the IP3 concentration.
We used immunoprecipitation and Western blots to measure the expression of IP3 receptor type 1 and type 2 in ventricles from wild-type and ob/ob mice, of which type 2 is the predominant isoform in cardiomyocytes (32). The mean expression of IP3 receptor type 2 was not different between ob/ob and wild-type ventricles (P = 0.41) (Fig. 3C). Moreover, there was no difference in the expression of IP3 receptor type 1 (data not shown).
The IP3 concentration was measured in wild-type and ob/ob cardiomyocytes in the absence and presence of insulin. Insulin had no effect on the IP3 concentration in wild-type cells, whereas it significantly (P < 0.05) increased the concentration by 30% in ob/ob cells (Fig. 3D).
Spontaneous Ca2+ waves.
Since IP3 has been shown to generate arrhythmias and spontaneous Ca2+ events in cardiomyocytes (17,19), we studied the occurrence of spontaneous propagating Ca2+ waves in resting (not paced) cardiomyocytes. Few spontaneous Ca2+ waves were observed under control conditions. Application of insulin (60 nmol/l) significantly increased the frequency of waves in both wild-type and ob/ob cells (Fig. 4A). The insulin-induced increase in the frequency of spontaneous waves was fully reversed after 15-min washout in both cell groups (data not shown). Pretreatment with wortmannin or 2-APB completely blocked the insulin-mediated increase in wave frequency (Fig. 4B).
The effect of IP3 was also tested under the same experimental conditions. IP3 application resulted in frequent spontaneous propagating Ca2+ waves, and the effect was completely blocked by 2-APB (Fig. 4B).
Mitochondrial Ca2+ transient.
Insulin had markedly different effects on cytosolic Ca2+ transients in wild-type and ob/ob cardiomyocytes, producing an increase in the amplitude in wild-type cells, whereas Ca2+ transients were broadened and extra transients occured during relaxation in ob/ob cells (see Table 1 and Fig. 2). Altered mitochondrial Ca2+ uptake in a beat-to-beat manner may have a role in this difference between wild-type and ob/ob cardiomyocytes, since mitochondria are known to contribute to the shaping of Ca2+ signals in cardiomyocytes (33) and diabetes is associated with impaired mitochondrial function (27). We therefore recorded transients of mitochondrial rhod-2 fluorescence at 1-Hz stimulation in the absence or presence of insulin (60 nmol/l). There was no difference between wild-type and ob/ob cells regarding the amplitude of mitochondrial Ca2+ transients under control conditions. However, the transients were significantly (P < 0.05) slower in ob/ob compared with wild-type cells (Fig. 5, top), with the time to peak and half-width being 182 ± 19 and 325 ± 24 ms in ob/ob cells (n = 14) vs. 115 ± 21 and 222 ± 20 ms in wild-type cells (n = 12), respectively. Application of insulin significantly increased the amplitude of mitochondrial Ca2+ transients in wild-type cells, whereas the amplitude was not changed in ob/ob cells (Fig. 5, bottom). Application of the IP3 analog also increased the amplitude in wild-type but not in ob/ob cells. Thus, the dynamic mitochondrial Ca2+ buffering during insulin or IP3 exposure was blunted in ob/ob cardiomyocytes.
DISCUSSION
Defects in the intracellular Ca2+ handling appear to be a generalized problem in diabetes (7). In the present study, we compared critical aspects of intracellular Ca2+ handling in ventricular cardiomyocytes of wild-type and ob/ob mice, a model of obesity and type 2 diabetes. The major novel results are 1) insulin triggers extra Ca2+ transients in ob/ob but not in wild-type cardiomyocytes, which indicates an increased susceptibility for developing arrhythmias in ob/ob hearts; 2) the effects of insulin on Ca2+ transients are reproduced by application of a membrane-permeant IP3 analog in ob/ob but not in wild-type cardiomyocytes; 3) in ob/ob cardiomyocytes, the effects of insulin and IP3 were blocked by 2-APB, and insulin increased the IP3 concentration; and 4) mitochondrial Ca2+ handling was impaired in ob/ob cardiomyocytes.
Ca2+ transients in obesity and type 2 diabetes.
Under control conditions, electrically evoked Ca2+ transients were smaller and slower in ob/ob cells, which may account for the decrease in peak contraction and slowed relaxation observed in different models of obesity and type 2 diabetes (34eC36). Moreover, a recent study on cardiomyocytes isolated from obese, type 2 diabetic mice lacking functional leptin receptors (db/db mice) also showed significantly smaller and slower Ca2+ transients in comparison with control cells (37).
Ca2+ transients in cardiomyocytes are mediated by Ca2+ influx through voltage-activated L-type Ca2+ channels, which activate sarcoplasmic reticulum Ca2+ release channels (ryanodine receptor-2) via a process known as Ca2+-induced Ca2+ release (38,39). Relaxation occurs when Ca2+ release is stopped and Ca2+ removed from the cytoplasm. This occurs predominantly by active reuptake into the sarcoplasmic reticulum by Ca2+ ATPase 2A, but Ca2+ extrusion out of the cell via Na+/Ca2+ exchange also contributes (39). The Ca2+ transient amplitude increased when insulin was applied in wild-type but not in ob/ob cardiomyocytes. This difference might reflect an inability of insulin to increase the L-type Ca2+ current in type 2 diabetes, whereas the inotropic effect of insulin is at least partly attributable to an increased L-type Ca2+ current in normal subjects (40eC42). This suggestion fits with our finding that 2-APB, which preferentially inhibits IP3-mediated signaling (43), had no effect on the insulin-induced increase in Ca2+ transient amplitude in wild-type cells, whereas it blocked the effects of insulin on Ca2+ handling in ob/ob cells. Thus, the insulin-induced increase of the Ca2+ transient amplitude in wild-type cells appears not to be mediated via IP3.
Possible role of IP3 in the insulin signaling.
Insulin application resulted in slowed Ca2+ transient kinetics and the appearance of frequent extra Ca2+ transients in ob/ob but not in wild-type cardiomyocytes. The insulin-mediated slowing of Ca2+ transients in ob/ob cells was due to an increased time to peak and slowed onset of the decay phase, whereas the rate of decline during the exponential decay phase was, if anything, increased (see Table 1). This indicates that insulin prolonged the sarcoplasmic reticulum Ca2+ release process whereas it had little effect on Ca2+ removal, which is dominated by the active sarcoplasmic reticulum Ca2+ reuptake (39). Application of a membrane-permeant IP3 analog gave results qualitatively the same as those of insulin in ob/ob cells; that is, there was no significant effect on the Ca2+ transient amplitude or the exponential decay rate, whereas the time to peak was increased, the early decay phase was slowed, and frequent extra Ca2+ transients were produced. Thus, both insulin and IP3 apparently increased the duration of action potential-mediated sarcoplasmic reticulum Ca2+ release in ob/ob cells, and this was accompanied by the triggering of extra Ca2+ transients. In accordance with these findings, insulin application caused an 30% increase in the IP3 concentration in ob/ob cardiomyocytes. On the other hand, the expression of type 1 and 2 IP3 receptors was not different between ob/ob and wild-type ventricles, and hence this cannot explain the differences between the two groups regarding the response to insulin and IP3.
The involvement of IP3 in insulin signaling in cardiomyocytes is further supported by the fact that the insulin- and IP3-mediated effects on electrically evoked Ca2+ transients in ob/ob cells were prevented by preincubation with 2-APB (see Fig. 3), one important action of which is to inhibit IP3 receptors (43). Furthermore, both insulin and IP3 induced spontaneous Ca2+ waves in rested wild-type and ob/ob cardiomyocytes, and this effect was fully blocked by 2-APB (Fig. 4). Finally, several studies have shown that IP3 can induce cardiac arrhythmias (17,18,19), which fits with the occurrence of insulin- and IP3-induced extra Ca2+ transients in ob/ob cells.
Possible role of defective mitochondrial function in impaired intracellular Ca2+ handling.
An increase in mitochondrial Ca2+ may stimulate oxidative metabolism via activation of enzymes involved in mitochondrial energy production (44eC46). Dynamic changes in mitochondrial Ca2+ are driven by the cytosolic Ca2+ transients in beating cardiomyocytes (31), thus providing a simple and elegant link between work and energy supply. Furthermore, a marked increase in mitochondrial Ca2+ in response to IP3-linked stimuli has been observed in a large variety of cell types (47). In terms of regulating global cytosolic Ca2+ handling, mitochondria are believed to act as a spatial buffering system that can blunt or slow propagating Ca2+ waves (48,49), as well as directly controlling Ca2+ release via IP3 receptors (49). Conversely, impaired mitochondrial Ca2+ accumulation may have deleterious effects by increasing cytosolic Ca2+ (49). In the present study, we showed slowed mitochondrial Ca2+ uptake in ob/ob cardiomyocytes compared with wild-type cells. Furthermore, the mitochondrial Ca2+ uptake did not increase in response to insulin or IP3 in ob/ob cells. Thus, the impaired mitochondrial Ca2+ uptake in ob/ob cells may contribute to the larger slowing of Ca2+ transients induced by insulin and IP3 in these cells, as well as the occurrence of extra Ca2+ transients.
We used rhod-2 to monitor mitochondrial Ca2+, and although this nonratiometric dye can readily measure transient changes in Ca2+, it is less suitable for detecting changes in basal mitochondrial Ca2+ accumulation. Thus, we are not able to distinguish between a basal mitochondrial Ca2+ overload or primary changes in mitochondrial Ca2+ flux kinetics as the major mechanism underlying the alterations observed in ob/ob cardiomyocytes.
Based on the present results, we propose the following model to explain the impaired Ca2+ handling in ob/ob cardiomyocytes: insulin increases the IP3 concentration in ob/ob cardiomyocytes, which prolongs electrically evoked sarcoplasmic reticulum Ca2+ release. Mitochondrial Ca2+ uptake is impaired in ob/ob cardiomyocytes, which decreases the ability to buffer the extra Ca2+ released during physiological challenges. Together, these defects in ob/ob cardiomyocytes cause a slowing of the Ca2+ transient and increase the probability of extra Ca2+ transients that may predispose for arrhythmias in vivo.
ACKNOWLEDGMENTS
This study was supported by the Swedish Research Council (project numbers 10842 and 14453), the Swedish Heart and Lung Foundation, Biovitrum Partner Fund, the Swedish Diabetes Foundation, and Funds at the Karolinska Institutet.
FOOTNOTES
A.K. and H.W. have received grant support from Biovitrum Partner.
Key Words: 2-APB, 2-aminoethoxydiphenyl borate CCE, capacitive calcium entry IP3, inositol 1,4,5-trisphosphate PI3K, phosphoinositide 3-kinase PLC, phospholipase C
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