Insulin and Inositol 1,4,5-Trisphosphate Trigger Abnormal Cytosolic Ca²⁺ Transients and Reveal Mitochondrial Ca²⁺ Handling Defects in Cardiomyocytes of ob/ob Mice

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.

Young (3–5 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 μmol · l⁻¹ · min⁻¹, 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.

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 μmol/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 40× 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 (3–6% 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 (ΔF) 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 (D_1/2), i.e., the duration at 50% of ΔF; and the time constant (τ) 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).

Rhod-2 was used to measure mitochondrial Ca²⁺ (31). Isolated cardiomyocytes were loaded with rhod-2 AM (10 μmol/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 μmol/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 (ΔF/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.

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).

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 μl 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 3–8% 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 peroxidase–conjugated 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).

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.

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 μmol/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.

We also tested the effect of a membrane-permeant IP₃ analog (10 μmol/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 μmol/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: ΔF/F₀ was 3.1 ± 0.5 without and 4.0 ± 0.3 with insulin, respectively (n = 7).

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).

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).

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.

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.

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 (34–36). 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 (40–42). 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.

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.

An increase in mitochondrial Ca²⁺ may stimulate oxidative metabolism via activation of enzymes involved in mitochondrial energy production (44–46). 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.

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.