Metallothionein Inhibits Myocardial Apoptosis in Copper-Deficient Mice: Role of Atrial Natriuretic Peptide

Abstract

SUMMARY:Dietary copper restriction causes heart hypertrophy in animal models. Several studies have indicated that this cardiomyopathy is mediated by oxidative stress. Metallothionein (MT), a low molecular weight and cysteine-rich protein, functions in protecting the heart from oxidative injury. We therefore used a cardiac-specific MT-overexpressing transgenic mouse model to test the hypothesis that MT inhibits copper deficiency-induced heart hypertrophy. Dams of both transgenic pups and non-transgenic littermates were fed a copper-adequate or copper-deficient diet, starting on the fourth day post-delivery, and the weanling mice were continued on the dams' diets until they were killed. Heart hypertrophy developed in copper-deficient pups by the fourth week of the combined pre-and post-weaning feeding and aggressively progressed until the end of the experiment (6 weeks). MT overexpression did not prevent the occurrence of heart hypertrophy, but inhibited the progression of this cardiomyopathy, which correlated with its suppression of cardiac lipid peroxidation. Corresponding to the progression of heart hypertrophy, myocardial apoptosis and atrial natriuretic peptide (ANP) production in the left ventricle were detected in non-transgenic copper-deficient mice; these effects were significantly suppressed in transgenic copper-deficient mice. Measurement of apoptosis by TUNEL assay and Annexin V-FITC confocal microscopy in primary cultures of cardiomyocytes revealed that ANP was largely responsible for the myocyte apoptosis and that MT inhibited ANP-induced apoptosis. The data clearly demonstrate that elevation of MT in the heart inhibits oxidative injury and suppresses the progression of heart hypertrophy in copper deficiency, although it does not block its initiation. The results suggest that MT inhibits the transition from heart hypertrophy to failure by suppressing apoptosis through inhibition of both cardiac ANP production and its apoptotic effect. (Lab Invest 2000, 80:745-757).

Molecular and cellular mechanisms of cardiotoxicity.

“…For example, we have observed that less than 0.5% of cells appeared apoptotic in myocardial tissue obtained from copper-deficient mice (49). At first glance, this number seems to be too insignificant to account for myocardial pathogenesis.…”

“…During myocardial pathologic remodeling, reexpression of ANP in myocytes of the left ventricle takes place (99)(100)(101). Recent studies have demonstrated that ANP is a factor responsible for myocardial apoptosis (49,102). Moreover, apoptosis plays a critical role in the development of heart failure (38-42,103,104).…”

“…Thus, the total cell loss can simply be accounted for by the rate of apoptosis plus necrosis. If apoptosis occurs at a constant rate of about 0.5% myocytes a day (49), the potential contribution of apoptosis to the overall loss of myocytes over a long period of time is significant.…”

Prevention of isoproterenol-induced cardiac hypertrophy by eugenol, an antioxidant

“…DNA fragmentation in isolated ventricular cells, indicative of apoptotic cell death, was detected in situ employing the terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay using a commercial kit (Roche Diagnostics, Mannheim, Germany) as described by Kang et al (8). Briefly, air-dried cell samples were fixed on glass slides with freshly prepared p -formaldehyde solution (4% in PBS, pH 7.4) for 1hour at 15 -25 0 C. The slides were then rinsed in PBS and incubated in a permeabilisation solution (0.1% Triton X-100, 0.1% sodium citrate) for 2 minutes on ice.…”

Catalase Protects Cardiomyocyte Function in Models of Type 1 and Type 2 Diabetes

“…We interpreted those results to mean that oxidative stress was essential for the development of diabetic cardiomyopathy. However, MT, in addition to being a powerful antioxidant protein, has other actions, including the binding of heavy metal ions, reducing apoptosis (12), and modulating transcription (13). Those other actions may have been critical to the protection afforded by MT.…”