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Metallothionein prolongs survival and antagonizes senescence-associated cardiomyocyte diastolic dysfunction: role of oxidative stress

Xiaoping Yang^*, Thomas A. Doser^*, Cindy X. Fang^*, Jennifer M. Nunn^*, Rajiv Janardhanan^*, Meijun Zhu, Nair Sreejayan, Mark T. Quinn and Jun Ren^*,1

^* Division of Pharmaceutical Sciences and Center for Cardiovascular Research and Alternative Medicine, and

Department of Animal Science, University of Wyoming, Laramie, Wyoming, USA; and

Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana, USA

¹Correspondence: Division of Pharmaceutical Sciences & Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, WY 82071-3375, USA. E-mail: jren{at}uwyo.edu

SPECIFIC AIMS

Senescence is accompanied by oxidative stress and cardiac dysfunction, although the link between the two remains unclear. This study examined the role of antioxidant metallothionein on cardiomyocyte function, superoxide generation, the oxidative stress biomarker aconitase activity, cytochrome c release, and expression of oxidative stress-related proteins, such as the GTPase RhoA and NADPH oxidase protein p47^phox in young (5–6 mo) and aged (26–28 mo) FVB wild-type and cardiac-specific metallothionein transgenic mice.

PRINCIPAL FINDINGS

1. Metallothionein mice showed longer life span than FVB mice evaluated by the Kaplan-Meier survival curve
The Kaplan-Meier survival curve shows that metallothionein transgenic mice display a 4 mo longer life span compared with FVB mice. The two curves start to separate from each other after 20 mo of age with metallothionein mice exhibiting a reduced mortality rate.

2. Aging-induced cardiac contractile defects were prevented by metallothionein
The mechanical and intracellular Ca²⁺ defects triggered by advanced age have been well documented but limited research focuses on single cardiomyocytes. We assessed mechanical properties of ventricular myocytes using a SoftEdge MyoCam system. The aging-induced prolongation of time-to-90% relengthening (TR₉₀) was corrected by expression of the metallothionein transgene (Fig. 1 ). To explore the possible role of intracellular Ca²⁺ homeostasis in aging and metallothionein-elicited response on cardiomyocyte mechanical function, we evaluated the intracellular Ca²⁺ transients in fura-2-loaded myocytes. Consistent with prolonged TR₉₀ in myocytes from aged FVB mice, myocytes from these mice displayed a significantly reduced rate of intracellular Ca²⁺ clearing, which was alleviated by metallothionein. Murine hearts contract at high frequencies. We examined the steady-state peak shortening amplitude under gradually increased stimulating frequency (0.1–5.0 Hz). The results demonstrate that peak shortening (PS) amplitude decreases dramatically with the increased stimulus frequency from 0.1 to 5.0 Hz. The degree of decline in peak shortening amplitude was significantly greater in the aged FVB group compared with young FVB mice, indicating decreased cardiac contractile reserve capacity at higher stress concentration under aging, while the aging-induced decline in peak shortening amplitude at high frequencies was blunted by expression of the metallothionein transgene.

View larger version (31K): [in this window] [in a new window]   Figure 1. Contractile properties of left ventricular myocytes from young (Y) and aged (O) FVB or metallothionein (MT) mice. A) Resting cell length. B) Peak shortening (PS, normalized to cell length). C) Maximal velocity of shortening (+dL/dt). D) Maximal velocity of relengthening (–dL/dt). E) Time-to-peak shortening (TPS). F) Time-to-90% relengthening (TR90). Mean ± SE; n = 140 cells/group; *P < 0.05 vs. FVB-Y group; #P < 0.05 vs. FVB-O group.

3. Aging-induced changes in oxidative stress and related protein biomarkers in the hearts were attenuated by metallothionein
Enhanced oxidative stress is commonly found with advanced age. Measurement of superoxide production in cardiomyocytes by DHE fluorescence microscopy showed myocytes from aged FVB mice produced significantly higher levels of superoxide as compared with myocytes from young mice, and this effect was attenuated by metallothionein. To investigate the intrinsic cause-consequence link between aging-induced oxidative stress and metallothionein over-expression, we detected aconitase activity, NADPH oxidase concentration, active RhoA, cytochrome c release using several techniques such as fluorescence, Western blotting, ELISA, and determination of enzyme activity. NADPH oxidase is the most predominant source of superoxide generation, leading to a variety of cardiovascular diseases including diabetes, hypertension, and metabolic syndrome. Similar to its effect on superoxide generation, metallothionein alleviated aging-induced up-regulation of p47^phox, suggesting a potential antagonistic effect of metallothionein on NADPH oxidase (Fig. 2 ). RhoA, a small GTPase, is well-documented as an inducer of oxidative stress. To explore the role of RhoA in aging-induced oxidative stress, active RhoA was determined by a pull-down assay. Not surprisingly, advanced age initiated an up-regulation of active RhoA, which was inhibited by expression of the metallothionein transgene. We also evaluated mitochondrial cytochrome c release as a marker for mitochondrial damage and found enhanced cytosolic cytochrome c levels in conjunction with reduced mitochondrial cytochrome c levels in aged FVB mouse hearts, indicating enhanced cytochrome c release. Nitrotyrosine is a biomarker for protein nitration by peroxynitrite. Evaluation of nitrotyrosine by ELISA indicated that neither aging nor metallothionein transgene expression affected nitrotyrosine formation, suggesting protein nitration is unlikely a major contributor to aging-induced cardiac defects. To further explore aging-induced oxidative stress, we measured cardiac aconitase levels. Aconitase is an iron-sulfur enzyme located in citric acid cycle, and the mitochondrial aconitase activity is closely associated with oxidative stress. Our results showed that aging led to decreased activity of aconitase, consistent with the previous studies, and metallothionein rectified the aging-induced decrease in aconitase activity.

View larger version (30K): [in this window] [in a new window]   Figure 2. Effect of metallothionein overexpression on p47phox expression in young and aged cardiomyocytes. Insets) Representative immunoblots of the NADPH oxidase subunit p47phox and ßbeta;-actin using specific antibodies. Mean ± SE; n = 3; *P < 0.05 vs. FVB-Y group; #P < 0.05 vs. FVB-O group.

CONCLUSIONS AND SIGNIFICANCE

Recent data suggested that metallothionein, a low molecular wieght heavy metal chelating antioxidant, exerts protective effects against diabetes- and insulin resistance-induced cardiac damage. However, the impact of metallothionein on cardiac aging and life span has not been elucidated.

Our present study revealed that metallothionein exerts similar effects against the aging process especially cardiac aging. Accumulation of oxygen free radicals or oxidative stress directly compromises ventricular function through NADPH oxidase activity, activation of stress signaling molecules, stimulation of renin-angiotensin system, and direct myogenic effects on heart muscles. Reduction of oxidative stress by either enzymatic or nonenzymatic antioxidants has been shown to improve cardiac function and reduce cardiac apoptosis. In the current study, advanced age elicited oxidative stress without hyperglycemia or hypertension, thus excluding contribution of diabetes and hypertension to our aging model. Data from this study revealed that ventricular myocytes from aged FVB mice displayed prolonged relaxation (TR₉₀), while all other mechanical indices (PS, maximal velocity of shortening/relengthening, time-to-peak shortening) were normal. Moreover, aged cardiomyocytes exhibited diminished stress tolerance manifested as significantly reduced peak shortening at higher stimulating frequencies. These data are consistent with our previous findings using murine model (at similar ages). Reduced intracellular Ca²⁺ clearance rate in aged FVB myocytes is in line with prolonged relaxation duration and reduced intracellular Ca²⁺ cycling ability (reduced PS at high stimulating frequencies) in cardiomyocytes from aged animals.

Given the facts that accumulation of oxygen free radicals or oxidative stress directly compromises ventricular function through NADPH oxidase activity, activation of stress signaling molecules, stimulation of renin-angiotensin system, and direct myogenic effects, our data suggest that metallothionein-elicited antagonism of aging-associated increase in oxidative stress may play a beneficial role against cardiac aging and prolong life span. Mitochondria have been speculated as the major site for oxidative stress-induced damage, although mammalian evidence is sparse. In present study, the aging-induced decrease of mitochondrial aconitase activity was alleviated by metallothionein showed us the possible role of metallothionein transgene. Recent evidence indicates that the small GTPase RhoA participates in the regulation of multiple cell functions, including adhesion, proliferation, migration, and Ca²⁺ homeostasis through kinase cascade activation. The current results indicated that aging-induce active RhoA increase was corrected by metallothionein.

Collectively, our present data revealed that over-expression of the metallothionein transgene prolonged life span (Fig. 3 ). Furthermore, the metallothionein transgene preserved cardiac contractile functions, improved intracellular Ca²⁺ homeostasis, and restored cardiac contractile capacity under high stimulus frequency, which are cardiac dysfunctions in aged mice. All these function alterations are associated with oxidative stress biomarkers. These results disclose that metallothionein may be beneficial in protecting against aging-induced cardiac dysfunction and oxidative stress and, thus, may prolong life span.

View larger version (20K): [in this window] [in a new window]   Figure 3. Scheme description of the metallothionein role in prolonged life span. Aging induces the increase of oxidative stress resulting cardiac dysfunctions. Over-expression of metallothionein, a potent free radical scavenger, prolongs the life span, and preserves the cardiac functions via alleviation of oxidative stress.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5288fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5288fjev1 20/7/1024    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yang, X. Articles by Ren, J. Search for Related Content PubMed PubMed Citation Articles by Yang, X. Articles by Ren, J.