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

Abstract Hypertension contributes to the high cardiac morbidity and mortality. Although oxidative stress plays an essential role in hypertensive heart diseases, the mechanism remains elusive. Transgenic mice with cardiac overexpression of metallothionein, a heavy metal‐binding scavenger, were challenged with NG‐nitro‐L‐arginine methyl ester (L‐NAME) for 14 days prior to measurement of myocardial contractile and intracellular Ca2+ anomalies as well as cell signalling mechanisms using Western blot and immunofluorescence analysis. L‐NAME challenge elicited hypertension, macrophage infiltration, oxidative stress, inflammation and cardiac dysfunction manifested as increased proinflammatory macrophage marker F4/80, interleukin‐1β (IL‐1β), intracellular O2- production, LV end systolic and diastolic diameters as well as depressed fractional shortening. L‐NAME treatment reduced mitochondrial membrane potential (MMP), impaired cardiomyocyte contractile and intracellular Ca2+ properties as evidenced by suppressed peak shortening, maximal velocity of shortening/relengthening, rise in intracellular Ca2+, along with elevated baseline and peak intracellular Ca2+. These unfavourable mechanical changes and decreased MMP (except blood pressure and macrophage infiltration) were alleviated by overexpression of metallothionein. Furthermore, the apoptosis markers including BAD, Bax, Caspase 9, Caspase 12 and cleaved Caspase 3 were up‐regulated while the anti‐apoptotic marker Bcl‐2 was decreased by L‐NAME treatment. Metallothionein transgene reversed L‐NAME‐induced changes in Bax, Bcl‐2, BAD phosphorylation, Caspase 9, Caspase 12 and cleaved Caspase 3. Our results suggest that metallothionein protects against L‐NAME‐induced myocardial contractile anomalies in part through inhibition of apoptosis.


| INTRODUC TI ON
Hypertension is a devastating cause of cardiovascular diseases which compromises cardiac structure and function. 1,2 It is well perceived that hypertension contributes to the ever rising risk of cardiovascular morbidity and mortality through onset and development of heart disease in addition to the well-established vascular anomalies. [2][3][4] A plethora of clinical and experimental studies has depicted the unique role of apoptosis in hypertensive heart disease. [5][6][7] Furthermore, reactive oxygen species (ROS) can directly regulate the apoptosis, mitochondrial integrity and survival in the hearts. 8 Indeed, antioxidant treatment has been shown to retard oxidative stress, mitochondrial injury and apoptotic cell death in pulmonary arterial hypertension-induced heart failure, suggesting the therapeutic value of antioxidants in treatment of cardiac diseases associated pulmonary hypertension. [9][10][11] It was also suggested that the mitochondria-targeted antioxidant peptide SS-31 alleviated mitochondrial oxidative injury and geometric hypertrophy in angiotensin II-induced hypertensive cardiomyopathy. 12 Therefore, manipulation of ROS through antioxidants and mitochondria may display some promises in the management of cardiac dysfunction in hypertensive heart disease. To this end, this study was designed to examine the impact of metallothionein in hypertension-induced changes of cardiac remodelling, inflammation, contractile function and cell apoptosis. Metallothioneins belong to group of low molecular weight intracellular cysteine-rich heavy metal-binding proteins. 13 Up-to-date, a variety of biological actions have been reported for metallothioneins including serving as potent antioxidants and free radical scavengers against reactive oxygen and nitrogen species including superoxide anions, hydrogen peroxide and peroxynitrite in pathological conditions such as obesity, cigarette smoking and diabetes. [14][15][16][17] In addition, data from our group suggested that metallothionein also preserved cardiac function via autophagy inhibition in cardiac dysfunction resulted from N G -nitro-L-arginine methyl ester hydrochloride (L-NAME)-induced hypertension. 18 Hence, the NO synthase inhibitor L-NAME was employed to generate experimental hypertensive model. 19,20 To discern the role of macrophage infiltration, inflammation, oxidative stress, apoptosis in metallothionein-and hypertensioninduced cardiac dysfunction, levels of macrophage infiltration marker F4/80, inflammation marker interleukin-1β (IL-1β), superoxide (O − 2 ) production and apoptotic markers Bax, bcl-2, phosphorylated BAD, Caspase 9 and Caspase 12 and cleaved Caspase 3 were evaluated in wild-type and metallothionein transgenic mice treated with L-NAME. Hypertension is associated with profound macrophage infiltration and inflammation, leading to cell and organ damage. 21,22 Furthermore, levels of Tenascin C and osteopontin-1 were monitored for any potential cardiac remodelling process.
JC-1 staining was employed to assess the mitochondrial membrane potential (MMP). TUNEL staining was used to assess apoptosis in hearts from FVB and metallothionein transgenic mice with or without L-NAME treatment.

| 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 cardiacspecific 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.

| Blood pressure and echocardiographic assessment
Conscious systolic and diastolic blood pressures were measured using a KODA semi-automated non-invasive blood pressure device

| 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

| 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

| 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

| 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.

| 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, and quantified by Quantity One software (Bio-Rad, version 4.6.2).
GAPDH was used as the loading control.

| 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.

| 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). 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 Ca 2+ decay (longer decay time) with little effect on baseline FFI levels.
Although metallothionein itself did not affect these intracellular Ca 2+ parameters, it rescued against L-NAME-induced changes in ΔFFI and intracellular Ca 2+ decay rate. In addition, L-NAME significantly promoted intracellular O − 2 production, the effect of which was obliterated by metallothionein (with little effect from metallothionein itself ( Figure 4D-E).

| Effect of metallothionein on L-NAME-induced mitochondrial damage
To explore the potential role of mitochondrial integrity in L-NAMEinduced 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 ΔΨm. These data demonstrated that the heavy metal scavenger alleviated L-NAME-induced mitochondrial injury.

| Effect of metallothionein on L-NAMEinduced apoptosis
To further evaluate if apoptosis plays a role in metallothioneinand 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-NAMEinduced 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

| 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 mac-  O − 2 production along with a drop of ΔΨm following L-NAME challenge, indicating a role for oxidative stress and mitochondrial injury in L-NAME-induced cardiac pathology. In addition, our data further revealed subtle although significant drop in SOD1 and HMOX-1 following L-NAME treatment, the effect of which was reconciled by metallothionein, denoting a role for antioxidant defence in L-NAMEand metallothionein-induced changes in mitochondrial function. In our hands, the F4/80 macrophage infiltration and elevated IL-1β levels were both noted following L-NAME treatment. This is in line with the previous report of overt inflammation in hearts from L-NAMEinduced hypertensive rats. 41 Interestingly, the heavy metal scavenger metallothionein only reconciled IL-1β levels without affecting Mean ± SEM, n = 4-5 mice per group, *P < 0.05 vs FVB group, #P < 0.05 vs FVB-L-NAME group alleviated cold stress-induced cardiac anomalies through attenuation of cardiac autophagy. 46,47 Our finding is consistent with earlier notion that antioxidants can attenuate cardiac dysfunction in hypertension. 9 Moreover, our recent study revealed that metallothionein can protect heart from L-NAME-induced cardiac dysfunction in experimental hypertension through autophagy regulation. 18 Our data shown here are in favour of the notion that hypertension may impair redox balance and mitochondria function in myocardium, which leads to apoptotic cell death, inflammation and cardiac dysfunction. It is noteworthy that cardiac-specific overexpression of metallothionein did not affect L-NAME-induced rises in both systolic and diastolic blood pressure, suggesting a relatively minor role of coronary endothelial metallothionein for blood pressure regulation although direct effect of vascular metallothionein cannot be ruled out.

| 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 Ca 2+ 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.

CO N FLI C T O F I NTE R E S T
The authors declared no conflict of interest for this work.

DATA AVA I L A B I L I T Y
All original data used in this work will be made available upon request.