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 N^G -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 ${\text{O}}_2^{-}$ 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.

Keywords: L-NAME; apoptosis; heart; hypertension; metallothionein.

Figure 1

Blood pressure in FVB and metallothionein (MT) mice with or without L‐NAME: A: Body weight; B: Systolic blood pressure; C: Diastolic blood pressure; and D: Mean blood pressure. Mean ± SEM, n = 10‐12 mice per group, *P < 0.05 vs FVB group

Figure 2

Effect of metallothionein (MT) on L‐NAME‐induced echocardiographic changes: A: Heart rate; B: LV posterior wall thickness during diastole (LVPWd): C: LV end systolic diameter (LVESD): D: LV end diastolic diameter (LVEDD); E: Fractional shortening; and F: LV mass (normalized to body weight). Mean ± SEM, n = 8‐10 mice per group, *P < 0.05 vs FVB group, #P < 0.05 vs FVB‐L‐NAME group

Figure 3

Effect of L‐NAME on cardiomyocyte contractile properties in FVB and MT mice: A: Resting cell length; B: Peak cell shortening (PS, normalized to resting cell length); C: Maximal velocity of cell shortening (+dL/dt); D: Maximal velocity of relengthening (−dL/dt); E: Time‐to‐peak shortening (TPS); and F: Time‐to‐90% relengthening (TR₉₀). Mean ± SEM, n = 78‐85 cells per group, *P < 0.05 vs FVB group, #P < 0.05 vs FVB‐L‐NAME group

Figure 4

Effect of L‐NAME on cardiomyocyte intracellular Ca²⁺ and ${\text{O}}_2^{-}$ properties in FVB and MT mice: A: Baseline fura‐2 fluorescence intensity (FFI); B: Rise in intracellular Ca²⁺ in response to electrical stimulus (ΔFFI); C: Intracellular Ca²⁺ decay rate; D: Representative intracellular fluorescent images depicting ${\text{O}}_2^{-}$ levels using DHE staining in four experimental groups; and E: Pooled data for ${\text{O}}_2^{-}$ production. Mean SEM, n = 52‐65 cells per group (panel A‐C) or n = 10 images per group for panel E, *P < 0.05 vs FVB group, #P < 0.05 vs FVB‐L‐NAME group

Figure 5

Effect of L‐NAME challenge on mitochondrial membrane potential measured using JC‐1 fluorescence: A‐D: Red fluorescence indicates hyperpolarized (J‐aggregate) mitochondria; E‐H: Green fluorescence indicates depolarized (monomer) mitochondria; I‐L: Merged red fluorescence and green fluorescence; and M. Quantification of ΔΨm expressed as ratio of aggregate/monomer fluorescence. Mean ± SEM, n = 20 cells per group, *P < 0.05 vs FVB group, #P < 0.05 vs FVB‐L‐NAME group

Figure 6

Confocal microscopic images depicting L‐NAME‐induced myocardial apoptosis: A‐P: Myocardial sections from WT and MT mice with or without L‐NAME treatment were stained with desmin (red), TUNEL (green) and nucleus with DAPI (blue). A‐D: Red fluorescence indicates cardiomyocytes; E‐H: Green fluorescence indicates apoptosis; I‐L: Blue fluorescence indicates nucleus; M‐P: are the merged images of the respective lines; Q, Quantitative analysis of apoptosis using TUNEL staining; and R: Caspase 3 activity. Mean ± SEM, n = 30 fields from 3 mice per group, *P < 0.05 vs FVB group, #P < 0.05 vs FVB‐L‐NAME group. DAPI = 4', 6‐diamidino‐2‐phenylindole; TUNEL = terminal dUTP nick end labelling

Figure 7

Western blot analysis of metallothionein (MT) and apoptotic markers in myocardium from FVB and MT mice treated with or without L‐NAME: A: Representative gel blots depicting expression of MT, BAD, phosphorylated BAD, Bcl‐2, Bax, Caspase 9, Caspase 12, cleaved Caspase 3 and GAPDH (used as the loading control); B: MT; C: Bax; D: p‐BAD‐to‐BAD ratio; E: BAD; F: Bcl‐2; G: Caspase 9; H: Caspase 12; and I: Cleaved Caspase 3. Mean ± SEM, n = 4‐5 mice per group, *P < 0.05 vs FVB group, #P < 0.05 vs FVB‐L‐NAME group

Figure 8

Western blot analysis of antioxidant and cardiac remodelling markers and immunofluorescent staining of proinflammatory macrophage in myocardium from FVB and metallothionein (MT) mice treated with or without L‐NAME: A: Representative gel blots depicting expression of IL‐1β, HMOX‐1, SOD1 and GAPDH (as the loading control); B: IL‐1β; C:HMOX‐1; D:SOD1; E: Representative images of immunofluorescent staining of myocardial macrophage with F4/80 antibody; F: Pooled data of F4/80 positive cells as a percentage of total cell number; G: Representative gel blots depicting expression of Tenascin C and Osteopontin‐1; H Tenascin C: and I: Osteopontin‐1. Mean ± SEM, n = 4‐5 mice per group, *P < 0.05 vs FVB group, #P < 0.05 vs FVB‐L‐NAME group