Cardiac-specific overexpression of metallothionein attenuates L-NAME-induced myocardial contractile anomalies and apoptosis

2.1 Experimental animals and hypertension model

All experimental procedures described here were approved by the Animal Care and Use Committees of the University of Wyoming (Laramie, WY, USA) and the Fourth Military Medical University (Xi'an, China). Transgenic mice with ~10-fold cardiac-specific overexpression of metallothionein (type IIa) were described in details in previous studies.23, 24 Five-to-6-month-old male transgenic mice and wild-type friend virus B (FVB) mice were provided 0.1 g/L N^G-Nitro-L-arginine Methyl Ester (L-NAME) in drinking water for 14 days to induce hypertension.19 Treatment of L-NAME for 14 days has been used routinely to induce experimental hypertension.25, 26 All mice were housed in a climate-controlled environment at 22.8 ± 2.0°C, 45%-50% humidity, under a 12/12-light/dark cycle with free access to food.

2.2 Blood pressure and echocardiographic assessment

Conscious systolic and diastolic blood pressures were measured using a KODA semi-automated non-invasive blood pressure device (Kent Scientific Corp, Torrington, CT, USA).27 Cardiac geometry and function were evaluated in anaesthetized (80 mg/kg ketamine and 12 mg/kg xylazine, ip) mice using two-dimensional guided M-mode echocardiography (Sonos 5500) equipped with a 15-6 MHz linear transducer. Left ventricular (LV) anterior and posterior wall dimensions during diastole (LVPWd) and systole (LVPWs) were recorded from three consecutive cycles in M-mode using methods adopted by the American Society of Echocardiography. Fractional shortening was calculated from LV end diastolic (LVEDD) and end systolic (LVESD) diameters according to the equation (LVEDD-LVESD)/LVEDD. Heart rates were averaged from 20 consecutive cycles.28

2.3 Isolation of murine cardiomyocytes

Cardiomyocytes were freshly isolated as described previously.29 After ketamine/xylazine sedation (ketamine 80 mg/kg and xylazine 12 mg/kg xylazine, ip), hearts were removed and perfused with Krebs-Henseleit bicarbonate (KHB) buffer at room temperature containing (in mM): 118 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 10 HEPES and 11.1 glucose. Solution was gassed with 5% CO₂/95% O₂. Hearts were isolated using liberase enzymatic digestion (Hoffmann-La Roche Inc, Indianapolis, IN, USA) for 20 minutes. Digested hearts were removed from the cannula and left ventricle was cut into small pieces. Undigested tissues were gently agitated and cell pellet was resuspended. Extracellular Ca²⁺ was added to a final concentration of 1.2 mmol/L gradually over a period of 30 minutes. Only rod-shaped myocytes with clear sarcomere striations were selected for mechanical properties and intracellular Ca²⁺ study.30

2.4 Cell shortening/relengthening

Mechanical properties of isolated cardiomyocytes were evaluated using an IonOptix™ soft-edge system (IonOptix, Milton, MA, USA). Cardiomyocytes were laid in a chamber mounted on the stage of an Olympus IX-70 inverted microscope and superfused (~2 mL/min at 25°C) with a Krebs-Henseleit bicarbonate buffer containing (in mmol/L) 131 NaCl, 4 KCl, 1 MgCl₂, 1 CaCl2, 10 HEPES and 10 glucose at pH 7.4. Myocytes were stimulated at a frequency of 0.5 Hz using a pair of platinum wires on the opposite sides of the chamber connected to a FHC stimulator (Brunswick, NE, USA). The cell was shown on the computer monitor by an IonOptix MyoCam camera. Cell shortening and relengthening were assessed by the following indices: resting cell length, peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR₉₀) and maximal velocities of shortening/relengthening (±dL/dt).28

2.5 Intracellular Ca²⁺ transients

Fura-2/AM (0.5 µmol/L) was added to a cohort of isolated cardiomyocytes for 10 minutes incubation and fluorescence intensity was measured with a dual-excitation fluorescence photomultiplier system (IonOptix). Cardiomyocyte was placed onto an Olympus IX-70 inverted microscope and imaged through a Fluor 40x oil objective. Cardiomyocytes were exposed to light emitted by a 75W lamp and passed through either a 360 or a 380 nm filter, while being stimulated to contract at a frequency of 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm and qualitative change in fura-2 fluorescence intensity (FFI) was deduced from the FFI ratio at the two wavelengths (360/380). Fluorescence decay time (single exponential) was calculated as an indicator of intracellular Ca²⁺ clearing.31

2.6 Intracellular fluorescence measurement of

Intracellular was monitored by changes in fluorescence intensity from intracellular probe oxidation. In brief, freshly isolated cardiomyocytes were loaded with 5 μmol/L dihydroethidium (DHE) (Molecular Probes, Eugene, OR) for 30 minutes at 37°C. Cells were evaluated using an Olympus BX-51 microscope with Olympus MagnaFire™ SP digital camera. Fluorescence was calibrated using InSpeck microspheres (Molecular Probes) and was quantitated using an ImagePro analysis software (Media Cybernetics, Silver Spring, MD).32

2.7 TUNEL staining

Frozen myocardial sections were obtained from the mid-section of the whole heart (about half way between apex and mitral valves) and were stained with a TUNEL staining kit (Roche). Sections were rinsed in PBS buffer for 10 minutes before incubation with proteinase K (20 μg/mL) in Tris-HCl for 30 minutes at 37°C. In a humidified chamber, sections were incubated with the anti-Desmin antibody (Cell Signaling, 1:50) at 4°C overnight. After that, sections were incubated with anti-Rabbit secondary conjugate with Alex Fluor 568 (Invitrogen) at 37°C for 1 hour. Sections were then incubated with the TUNEL staining solution at 37°C for 1 hour in a humidified chamber. Following PBS wash, myocardial sections were stained with DAPI for 10 minutes at 37°C in a humidified chamber. Sections were mounted with 50% glycerol and were used for fluorescent detection using confocal microscopy. The percentage of apoptotic cells in total cells was calculated.33, 34

2.8 Immunofluorescent staining of myocardial tissues

The anti-F4/80 antibody was used to label macrophages to examine the inflammatory response triggered by L-NAME. In brief, frozen myocardial sections (mid-section from the whole heart) were gently mixed in PBS buffer for 10 minutes and then incubated in proteinase K (20 μg/mL) solution at 37°C for 30 minutes. Goat serum was added for blocking at 37°C for 1 hour. After rinsing in PBS, samples were incubated with anti-F4/80 (1:100) antibody at 4°C overnight. To remove the excess primary antibody, sections were washed in PBS three times for 10 minutes each. Samples were then incubated with Alex Fluor 568 secondary antibody for 2 hour at room temperature. Cellular nuclei were stained with DAPI (10 μg/mL) for 15 minutes at room temperature. Slides were finally imaged using a confocal microscopy and the F4/8-positive cells were expressed as a percentage of total cell numbers.35

2.9 Measurement of mitochondrial membrane potential (ΔΨm)

Given that mitochondrial function is key to cardiomyocyte viability, the cationic lipophilic probe JC-1 was employed to measure ΔΨm. The dynamic change of ΔΨm was displayed by change in the ratio between red (aggregated JC-1) and green (monomeric JC-1) fluorescence. Murine cardiomyocytes (with a viability of 75%-80%) were suspended in HEPES-saline buffer and mitochondrial membrane potential (ΔΨm) was detected as described. Briefly, following a 10-min preincubation with 5 μmol/L JC-1 at 37°C, cells were rinsed twice by sedimentation using HEPES buffer free of JC-1. The cardiomyocytes were observed by a confocal microscopy. Fluorescence was read at excitation wavelength of 490 nm and emission wavelength of 530 and 590 nm using a spectrofluorimeter (SpectraMaxGeminiXS, Spectra Max, Atlanta, GA, USA). Results in fluorescence intensity were expressed as 590 to 530 nm emission ratio. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 μmol/L) was employed as a positive control for mitochondrial membrane potential.36, 37

2.10 Caspase-3 activity assay

Caspase 3 activity was measured according to the previously published method.31, 33 In Brief, 1 mL of PBS was added to whole heart tissues. Then the tissues were homogenized and centrifuged at 10 000 × g for 10 minutes at 4°C. The supernatant was discarded and pellets were lysed in 100 μL of ice-cold lysis buffer (50 mmol/L HEPES, pH 7.4, 0.1% CHAPS, 1 mmol/L dithiothreitol (DTT), 0.1 mmol/L EDTA, 0.1% NP40). The assay for Caspase 3 activity was carried out in a 96-well plate. Each well contained 30 μL of lysate, 70 μL of assay buffer (50 mmol/L HEPES,pH 7.4, 0.1% CHAPS, 100 mmol/L NaCl, 10 mmol/L DTT and 1 mmol/L EDTA) and additional 20 μL of Caspase 3 colorimetric substrate Ac-DEVD-pNA. The 96-well plate was incubated at 37 ^◦C for 2 hours, during the time Caspase in the sample was allowed to cleave the chromospheres pNA from the substrate molecule. Absorbance readings were obtained at 405 nm with the Caspase 3 activity being directly proportional to the colorimetric reaction. Protein concentration was determined using the Bradford method. The final Caspase 3 activity was expressed as picomoles of pNA released per microgram of protein per minute.33, 38

2.11 Western blot analysis

Whole heart protein samples containing equal amount of proteins were loaded on 10% SDS-polyacrylamide gels in a minigel apparatus (Mini-PROTEAN II, Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose membranes (0.2 μm pore size, Bio-Rad). The membranes were blocked with 5% milk in TBS-T for 2 hours and were incubated overnight at 4°C with anti-metallothionein, anti-BAX, anti-phospho-BAD, anti-BAD, anti-Bcl2, anti-Caspase 9, anti-Caspase 12, anti-cleaved Caspase 3, anti-tenascin C, anti-osteopontin-1, anti-IL-1β, anti- copper/zinc superoxide dismutase-1 (SOD1), anti-heme oxygenase (HMOX-1) (SOD1 and HMOX-1 were used to assess antioxidant capacity) and anti-GAPDH (loading control) antibodies. After incubation and washing steps, the secondary antibodies conjugated with horseradish peroxidase (HRP) were added. The proteins were detected by enhanced chemiluminescence (Amersham Pharmacia, Piscataway, NJ, USA) and quantified by Quantity One software (Bio-Rad, version 4.6.2). GAPDH was used as the loading control.

2.12 Statistical analysis

All statistical analysis was performed with GraphPad Prism 4.0 software. Data were expressed as Mean ± SEM. Statistical significance (P < 0.05) was estimated by one-way analysis of variance (ANOVA) followed by a Turkey's test for post hoc analysis.

3.1 General biometric features, blood pressure and echocardiographic properties

Our data shown in Figure 1 revealed that treatment of 0.1 g/L L-NAME in drinking water for 14 days significantly increased diastolic, systolic and mean blood pressures in both FVB and MT mice, in a comparable manner. Cardiac overexpression of metallothionein alone did not affect blood pressures. Neither L-NAME treatment nor metallothionein transgene affected body weight. As demonstrated in Figure 2, echocardiographic assessment suggested that L-NAME treatment dramatically increased LV end systolic diameter (LVESD) and LV end diastolic diameter (LVEDD) accompanied with diminished fractional shortening, the effect of which was negated by metallothionein transgene. Metallothionein transgene itself did not display any detectable effect on these echocardiographic parameters. Neither L-NAME treatment nor metallothionein, or both, affected heart rate, LV wall thickness and LV mass (normalized to body weight).

3.2 Effect of metallothionein on L-NAME-induced changes in cardiomyocyte contractile and intracellular Ca²⁺ properties as well as intracellular production

Neither L-NAME treatment nor metallothionein transgene exhibited any overt effect on resting cardiomyocyte cell length. Fourteen days of L-NAME treatment significantly decreased peak shortening and maximal velocity of shortening/relengthening (±dL/dt) as well as prolonged time-to-90% relengthening (TR₉₀) without affecting time-to-PS (TPS). Although metallothionein itself failed to affect these cardiomyocyte mechanical indices, it abrogated L-NAME-induced mechanical alterations (Figure 3). To further elucidate the possible mechanisms underlying L-NAME- and metallothionein-induced cardiac mechanical changes, intracellular Ca²⁺ and intracellular were measured using the intracellular fluorescent dyes Fura-2 and DHE respectively. Data presented in Figure 4A-C displayed that L-NAME treatment significantly suppressed electrically stimulated rise in fura-2 fluorescence intensity (ΔFFI) and increased intracellular Ca²⁺ decay (longer decay time) with little effect on baseline FFI levels. Although metallothionein itself did not affect these intracellular Ca²⁺ parameters, it rescued against L-NAME-induced changes in ΔFFI and intracellular Ca²⁺ decay rate. In addition, L-NAME significantly promoted intracellular production, the effect of which was obliterated by metallothionein (with little effect from metallothionein itself (Figure 4D-E).

3.3 Effect of metallothionein on L-NAME-induced mitochondrial damage

To explore the potential role of mitochondrial integrity in L-NAME-induced cardiac dysfunction, the cationic lipophilic probe JC-1 was employed to monitor mitochondrial membrane potential (ΔΨm) in cardiomyocytes from FVB and metallothionein mice with or without L-NAME treatment. Alteration of ΔΨm was reflected by changes of ratio between red (aggregated JC-1) and green (monomeric form of JC-1) fluorescence. Results shown in Figure 5 depicted an overt reduction in the ratio between red and green fluorescence in response to L-NAME challenge, illustrating a drop in ΔΨm which represents mitochondrial injury. Intriguingly, the L-NAME-induced reduction of mitochondrial ΔΨm was ablated by the heavy metal scavenger while metallothionein alone fails to exhibit any effect on ΔΨm. These data demonstrated that the heavy metal scavenger alleviated L-NAME-induced mitochondrial injury.

3.4 Effect of metallothionein on L-NAME-induced apoptosis

To further evaluate if apoptosis plays a role in metallothionein- and L-NAME-induced changes in myocardial function, apoptosis was examined in myocardium from FVB and metallothionein transgenic mice using TUNEL staining, Caspase 3 assay and Western blot. TUNEL staining revealed a greater percentage of TUNEL-positive cells in L-NAME-induced FVB mice, the effect of which was ablated by metallothionein with little notable effect from the transgene itself. These findings were consolidated by Caspase 3 activity assay where metallothionein nullified L-NAME-induced rise in Caspase 3 activity (Figure 6). Our results further demonstrated that the level of cleaved Caspase 3 was increased in L-NAME-induced FVB mice while metallothionein overexpression significantly attenuated the up-regulation of cleaved Caspase 3 (Figure 7H). Results of Western blot shown in Figure 7A-B also revealed that L-NAME treatment did not affect the levels of metallothionein in either FVB or MT mice. MT overexpression was verified using Western blot analysis. However, L-NAME challenge overtly promoted levels of the pro-apoptotic proteins BAX, Bad (reduced phosphorylation), Caspase 9 and Caspase 12 as well as down-regulated anti-apoptotic protein Bcl-2. Consistent with the findings from TUNEL staining and Caspase 3 activity assays, metallothionein transgene protected against L-NAME-induced changes in these apoptotic protein markers without eliciting any effects by itself (Figure 7A-H).

3.5 Effects of metallothionein on L-NAME-induced cardiac remodelling and inflammation

To examine if the protective role of metallothionein against L-NAME-induced cardiac dysfunction is related to antioxidant capacity, cardiac remodelling, macrophage infiltration and inflammation, levels of antioxidant proteins SOD1 and HMOX-1, remodelling protein markers tenascin C and osteopontin-1 and proinflammatory maker IL-1β were monitored using Western blot analysis, while macrophage infiltration was evaluated by immunohistochemistry. Our results from immunoblot and immunofluorescent staining indicated that L-NAME led to elevated level of IL-1β, reduced expression of antioxidant HMOX-1 and SOD1, without affecting the levels of remodelling protein markers tenascin C and osteopontin-1. Although metallothionein itself did not elicit any response on these protein markers, it reversed L-NAME-induced changes in IL-1β, SOD1 and HMOX-1 with little effect on the remodelling protein markers (Figure 8A-D and G-I). Interestingly, results of immunofluorescent staining using F4/80 antibody showed that L-NAME challenge triggered pronounced macrophage infiltration in a comparable manner in both FVB and metallothionein mice (P < 0.05 for L-NAME-treated groups vs FVB group). Metallothionein itself did not affect macrophage infiltration (Figure 8E-F).

4.1 Experimental limitations

Although our data suggest that metallothionein rescues against L-NAME challenge-induced cardiac functional anomalies, several limitations exist. First and perhaps foremost, expression of metallothionein (type IIa) is non-physiological and may elicit off-target effects, making it difficult to evaluate the translational efficacy of this work. Second, the 14-day L-NAME-induced hypertension is somewhat “artificial” and may not recapitulate the genuine pathological changes in hypertensive heart disease derived from chronic essential hypertension. For example, no notable cardiac remodelling was noted in light of the overt presence of macrophages. Thus, special caution should be taken in data interpretation and extrapolation to hypertensive heart diseases.

In summary, our results revealed that the antioxidant metallothionein rescued against cardiac contractile and intracellular Ca²⁺ derangement, inflammation, loss of redox balance (superoxide production and antioxidant defence), mitochondrial injury and apoptosis in a L-NAME-induced experimental hypertension model. Given the pivotal role of antioxidants in the regulation of cardiac structure and function, our data favour a role for antioxidants in the management of hypertension-associated cardiac dysfunction.