Metallothionein Prevents Diabetes-Induced Deficits in Cardiomyocytes by Inhibiting Reactive Oxygen Species Production

OVE26 diabetic mice have been previously described (17). MT mice that overexpress MT 10-fold in the heart were also previously described (18). Transgenic mice of both lines were identified by the presence of phenotypic markers. OVE26-positive mice were recognized by the presence of small eyes as a result of the co-integration of the GR19 gene, which is expressed in the eye. MT-positive mice were recognized by the presence of coat color as a result of co-integration of the tyrosinase gene that corrects the mutant tyrosinase gene of FVB mice (19). Age-matched OVE26, OVE26MT, and FVB mice of both sexes were obtained at 20 weeks of age. All transgenic and nontransgenic animals were maintained on the inbred FVB background. Mice were maintained on a 12-h light/dark cycle and received food (Purina Laboratory Rodent Diet 5001) and water ad libitum. Serum triglycerides were measured with a Sigma triglyceride (UV) kit. The U.S. Department of Agriculture certified institutional animal care committee approved all animal procedures.

Single ventricularmyocytes were isolated by enzymatic dissociation with collagenase II (Worthington, NJ) from adult mice by modification of described procedures (4). Briefly, animals received an injection of heparin (1,000 units/kg, i.p.) and were anesthetized with ketamine (150 mg/kg, i.p.) and xylazine (22.5 mg/kg, i.p.). Their hearts were rapidly removed and perfused (at 37°C) with oxygenated (5% CO₂/95% O₂) Krebs-Henseleit (KH) calcium-free buffer (in mmol: 135 NaCl, 4.0 KCl, 1.0 MgCl₂, 0.33 NaH₂PO₄, 10 HEPES and glucose, and 10 Butanedione [pH 7.4]). Hearts were then perfused with the same Ca²⁺-free KH buffer with collagenase II (0.9 mg/ml) for 15–20 min until the heart became flaccid. After perfusion, the left ventricle was removed, minced, and resuspended with same Ca²⁺-free KH at room temperature. Extracellular Ca²⁺ was added incrementally back to 1.2 mmol/l. Isolated myocytes were maintained at room temperature in a serum-free medium consisting of medium 199 (GIBCO) with Hanks’ salts containing 25 mmol HEPES for additional mechanical and fluorescent studies. Mechanical properties remained relatively stable in myocytes for 8–10 h in the serum-free medium. Cell yield was ∼50–70%. There was no notable difference of yield between normal FVB and transgenic mice. Myocytes with obvious sarcolemmal blebs or spontaneous contractions were not used. Only rod-shaped myocytes with clear edges were used for recording of mechanical properties, intracellular Ca²⁺ transients, and ROS fluorescent measurement.

Mechanical properties of ventricular myocytes were assessed using a video-based edge-detection system (IonOptix, Milton, MA) (4). Cells were placed in a Teflon glass coverslip dish (Harvard Apparatus, Holliston, MA) mounted on the stage of an inverted microscope (Olympus, IX-70). The cells were field-stimulated at a frequency of 1.0 Hz, 4-ms duration, using a pair of platinum wires placed on opposite sides of the dish chamber connected to the MyoPacer Field Stimulator (IonOptix). The polarity of the stimulatory electrodes was reversed frequently to avoid possible build-up of electrolyte by-products. The myocytes being studied were displayed on the computer monitor using an IonOptix MyoCam camera, which rapidly scans the image area every 8.3 ms such that the amplitude and velocity of shortening/relengthening was recorded with good fidelity. The soft-edge software (IonOptix) was used to capture changes in cell length during shortening and relengthening. Cell shortening and relengthening were assessed using the following indexes: peak shortening (PS), time to 90% PS (TPS₉₀), time to 90% relengthening (TR₉₀), and maximal velocities of shortening and relengthening (±dL/dt).

A separate cohort of myocytes was loaded with fura-2/AM (1.0 μmol/l) for 30 min, and fluorescence measurements were recorded with a dual-excitation fluorescence photomultiplier system (IonOptix). Myocytes were placed in a dish chamber on the stage of an Olympus IX-70 inverted microscope and imaged through a Fluor 100× oil objective. Cells were exposed to light emitted by a 75-W lamp and passed through either a 360- or a 380-nm filter (bandwidths were ±15 nm), while being stimulated to contract at 1.0 Hz. Fluorescence emissions were detected between 480 and 520 nm by a photomultiplier tube after first illuminating the cells at 360 nm for 0.5 s then at 380 nm for the duration of the recording protocol (333 Hz sampling rate). The 360-nm excitation scan was repeated at the end of the protocol, and qualitative changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) were inferred from the ratio. Intracellular Ca²⁺ transients were measured as changes in fura2 fluorescence intensity (FFI). ΔFFI was determined as the difference between the levels of Ca²⁺ in the systolic and diastolic conditions (ΔFFI = peak FFI − baseline FFI). The time course of the fluorescence signal decay (τ: the duration where Ca²⁺ transient decays 67% from the peak level) was calculated to assess intracellular Ca²⁺ clearing rate.

The membrane-permeable probe 5-(6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) enters cardiomyocytes and produces a fluorescent signal after intracellular oxidation by ROS such as hydrogen peroxide and hydroxyl radical (20). Intracellular oxidant stress was monitored by measuring changes in fluorescence resulting from intracellular probe oxidation. Isolated myocytes were loaded with 1 μmol/l CM-H₂DCFDA (Molecular Probes, Eugene, OR) for 30 min. After myocytes were washed, fluorescence intensity from individual cells was measured using an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Cells were sampled at random in each preparation using an Olympus IX70 inverted microscope equipped with a digital cooled charged-coupled device camera and ImagePro image analysis software (Media Cybernetics, Silver Spring, MD). Fluorescence was calibrated with InSpeck microspheres (Molecular Probes). Calibration curves were generated, and cell brightness was measured. The InSpeck microspheres were used as internal standards. The intensities of cells and beads were calibrated by subtraction of the background, and the final result for comparison was assessed by ratio of myocyte fluorescent intensities divided by intensities of internal fluorescent beads. The cardiomyocytes from each group were incubated at room temperature separately in M199 medium with normal glucose concentration as control (5.5 mmol/l), high glucose (25 mmol/l), normal glucose plus angiotensin II (50 nmol/l), and normal glucose plus hydrogen peroxide as a positive control (H₂O₂, 40 μmol/l). Some incubations included 1 μmol/l diphenyleneiodonium (DPI) or 0.1 μmol/l losartan. The different culture conditions were begun 30 min before CM-H₂DCFDA was added to the medium. After 30 min of incubation of CM-H₂DCFDA, the myocytes were washed twice and images were captured by Olympus IX70 inverted microscope equipped with a charged-coupled device camera. For results in Fig. 5, images were captured from live cells. For results in Fig. 6, myocytes were fixed in 4% paraformaldehyde after incubation and stored at 4°C for up to 16 h before imaging. There were 100 cells in each group.

Data are expressed as means ± SE. Statistical comparisons were performed by ANOVA and Student-Newman-Keuls post hoc tests among different groups. Significance was defined as P < 0.05.

Similar rod-shaped cardiomyocytes with good striation and edges were obtained from FVB, OVE26, and OVE26MT mice. The averaged resting myocyte length was not significantly different among the two transgenic and the wild-type mouse groups (OVE26 113.13 ± 2.83 μm, OVE26MT 118.44 ± 2.72 μm, and FVB 121.09 ± 3.04 μm). As shown in Figs. 1 and 2, cardiomyocyte contractile function from OVE26 mice was markedly depressed. The cardiomyocytes from OVE26 mice showed significantly reduced peak shortening (OVE26 versus FVB, P < 0.01; Fig. 3B) and ±dL/dt_max (OVE26 versus FVB, P < 0.01; Fig. 2A and B) as compared with those from FVB mice. Myocytes from OVE26 mice also exhibited significantly prolonged TPS₉₀ (OVE26 versus FVB, P < 0.05; Fig. 1C) and TR₉₀ (OVE26 versus FVB, P < 0.01; Fig. 1D). Overexpressed MT in the heart significantly reversed the impaired myocyte contractile performance (PS, Fig. 1B; ±dL/dt_max, Fig. 2A and B) and prolongation of myocyte contraction and relengthening (TPS₉₀ and TR₉₀, Fig. 1C and D) by diabetes (OVE26MT versus OVE26, P < 0.01). The benefits provided by MT overexpression occurred despite the fact that MT does not reduce diabetic blood glucose, as we previously reported (16), or elevated serum triglyceride levels (OVE26 349.9 ± 62.9, OVE26MT 467.7 ± 29.5, and FVB 107.6 ± 7.7, P < 0.001 for FVB versus both other groups by ANOVA and Bonferroni test).

Myocytes from the three groups exhibited similar peak and baseline FFI (data not shown) and no differences of ΔFFI (Fig. 3B). As shown in Fig. 3C, recovery of the Ca²⁺ transient occurred more rapidly in control FVB cardiomyocytes than in OVE26 myocytes (τ, FVB 178.78 ± 8.77 ms vs. OVE26 261.97 ± 14.23; P < 0.01). However, overexpressed MT significantly facilitated the calcium-clearing rate in diabetic cardiomyocytes (OVE26MT 190.34 ± 10.26 ms vs. OVE26 261.97 ± 14.23 ms; P < 0.01).

Figures 4 and 5 show ROS production measured with CM-H2DCFDA. Normal extracellular glucose produced similar ROS intensities among the cardiomyocytes from all three groups. After exposure of myocytes to 40 μmol/l H₂O₂, the fluorescent intensities were significantly increased in the cardiomyocytes from the three groups compared with normal glucose incubation, but there were no significant differences among the three groups. Treatment of myocytes with high glucose (25 mmol/l) or angiotensin II (50 nmol/l) for 60 min did not affect the ROS level in control cardiomyocytes but caused a significantly increased ROS in the OVE26 cardiomyocytes compared with those in the normal condition of glucose (OVE26 HG versus OVE26 NG, P < 0.001). However, the effects of high glucose or angiotensin II on ROS production were completely abolished by overexpressed MT (OVE26MT versus OVE26, P < 0.001).

To ascertain the source of increased ROS in diabetic cells, we incubated OVE26 and FVB myocytes with 10 μmol/l DPI and 0.1 μmol/l losartan. DPI is an inhibitor of flavoprotein-containing enzymes, including NADPH oxidase. Losartan is a specific angiotensin II type I receptor antagonist. As shown in Fig. 6, both DPI and losartan eliminated the increased fluorescence induced in OVE26 myocytes by either angiotensin II or high glucose. These inhibitors had no significant effect on fluorescence in FVB myocytes.

This is the first study to evaluate the efficacy of permanent antioxidant protection of individual cardiomyocytes from diabetes. These studies were carried out in myocytes from the OVE26 mouse. Although this model has not been widely used to study diabetes complications, it provides several advantages: all OVE26 mice have a very consistent onset and severity of diabetes, their duration of diabetes is long, they can live with hyperglycemia for 2 years without insulin therapy, and specific damage to the β-cell occurs without use of toxic drugs. Diabetes is produced in heterozygous OVE26 animals, allowing these mice to be cross-bred easily to other strains of mice (16).

OVE26 diabetic cardiomyocytes had reduced contractility and prolonged calcium transients. The amplitude of cell shortening was decreased, the duration of contraction was increased, and the rates of contraction and relaxation were reduced. This demonstrates that the impaired contractility of the intact diabetic heart is due to defects in the individual myocyte, rather than just increased cardiac fibrosis. These results are in agreement with data obtained in other models of diabetes (21,22) and confirm the validity of the OVE26 model.

Calcium transients in diabetic cardiomyocytes were also abnormal. As seen in other diabetic models (22), decay of intracellular Ca²⁺ transients in OVE26 myocytes was significantly slowed. The slow Ca²⁺ clearing is consistent with and may be the cause of the prolonged duration of myocyte relengthening. Slow relengthening is a hallmark of diabetic cardiomyopathy. In contrast to decay rate, resting and peak Ca²⁺ levels were similar between the control and diabetic cardiomyocytes. Reports regarding intracellular Ca²⁺ level in diabetic cardiomyocytes are somewhat controversial. Early studies reported an increase in resting Ca²⁺ (23), whereas Noda et al. (24) reported a reduced basal Ca²⁺ in diabetic cardiomyocytes. Several reports demonstrated no differences in basal Ca²⁺ or peak Ca²⁺ levels between diabetic and control cardiomyocytes (25,26,27), which is consistent with our results. Therefore, there may not be significant alteration of Ca²⁺ levels that correspond to the depressed contraction in diabetic cardiomyocytes. Attenuated calcium sensitivity of the contractile apparatus or changes in cardiac contractile proteins may be more important than absolute Ca²⁺ levels to the reduced shortening of diabetic cardiomyocytes (28).

Many studies report that diabetic hearts exhibit characteristics of oxidative stress. OVE26 hearts have increased levels of oxidized glutathione (16), and hearts of BB (29) and streptozotocin (30) diabetic rats show induction of antioxidant enzymes. This ROS stress of diabetes can produce oxidative damage including lipid peroxidation (31,32) and nitrotyrosine formation (20). To determine whether oxidative stress was a cause or consequence of diabetic cardiomyopathy, we placed the cardiac transgene for the antioxidant protein MT on the OVE26 background (16). MT overexpression was remarkably effective in ameliorating functional deficits in OVE26 diabetic cardiomyocytes, despite that MT overexpression in no way reduced the exposure of cardiomyocytes to the severe hyperglycemic environment in these mice. MT overexpression completely reversed diabetes-induced diminished PS amplitude, prolonged duration and reduced maximal velocities of contraction and relaxation, and the slower intracellular Ca²⁺ transient decay. This indicates that ROS is essential to the pathogenesis of diabetic cardiomyopathy. Our preliminary data showing that two other antioxidant transgenes (unpublished results) protect from OVE26 diabetic cardiomyopathy confirm that it was the antioxidant actions of MT that protected the diabetic heart.

Increased oxidative damage suggests elevated ROS production in diabetic cardiomyocytes. Consistent with this, OVE26 myocytes produced more ROS in the presence of angiotensin II than myocytes from control mice or mice protected by the MT transgene. Bendall et al. (33) recently showed that angiotensin II stimulates cardiac NADPH oxidase, an enzyme that produces superoxide at the expense of the electron donor NADPH. Losartan blocked the response to angiotensin II in OVE26 myocytes. Consistent with a possible role for NADPH oxidase DPI, an inhibitor of flavoprotein enzymes such as NADPH oxidase blocked excess ROS production produced by angiotensin II.

High glucose also stimulated ROS production in OVE26 myocytes. Several sources have been proposed for enhanced ROS formation in hyperglycemia. These include mitochondrial electron transport, NADPH oxidase, and autoxidation of glucose. Mitochondria are the major source of ROS in most cells as a result of superoxide-generating electron-transport intermediates such as ubisemiquinone. Increased substrate for electron transport increases the mitochondrial membrane potential, which results in increased ROS production (34). The Brownlee laboratory has shown that hyperglycemia increases electron transport‐generated superoxide in endothelial cells, which activates several pathways thought to lead to complications of diabetes (35). Glucose autoxidation and protein glycation both are processes that are accelerated by high glucose and produce superoxide radical or other ROS (36,37). NADPH oxidase has been shown to be stimulated by hyperglycemia in several cell types (38,39), and its mRNA in endothelial cells is induced sevenfold by high glucose (39). High glucose can turn on NADPH oxidase by de novo synthesis of diacylglycerol (40), which activates PKC, which in turn can activate NAD(P)H oxidase by phosphorylation of p47(phox) (41).

The response of OVE26 cardiomyocytes to high glucose was similar to their response to angiotensin II. Glucose, like angiotensin II, increased ROS levels only in diabetic cardiomyocytes, and in both cases the response was prevented by DPI, MT overexpression, and losartan. This similarity suggests that high glucose acts via angiotensin II or that high glucose requires the simultaneous activation of the angiotensin II system. That losartan and DPI blocked the high glucose response suggests involvement of NADPH oxidase. However, the DPI results cannot be considered a definitive indication for the involvement of NADPH oxidase, because DPI inhibits other flavoprotein-containing enzymes that may play a role in ROS production. This includes inhibition of complex II of the mitochondrial electron transport chain.

In summary, our data demonstrate that 5 months of diabetes resulted in OVE26 cardiomyocytes with reduced mechanical performance and slow intracellular Ca²⁺ clearing. Permanent antioxidant treatment by MT overexpression prevents these defects. OVE26 myocytes exhibited a significantly greater ROS response to high glucose or angiotensin II, and this was also prevented by MT overexpression. Acute treatment with DPI or losartan blocked the enhanced diabetic ROS production, suggesting that high glucose requires angiotensin II activity and that ROS are produced by a flavoprotein-containing enzyme. Chronic ROS elevation in diabetic cardiomyocytes damages contractility, impairs calcium handling, and may predispose the myocyte to produce greater levels of ROS.

This work was supported by grants from the National Institutes of Health (HL62892, HL6677) and the Commonwealth of Kentucky Research Challenge Trust Fund.