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.
Highlights
To further evaluate if apoptosis plays a role in metallothionein‐ and L‐NAME‐induced changes in myocardial function, apoptosis was examined in myocardium from friend virus B (FVB) and metallothionein transgenic mice using TUNEL staining, Caspase 3 assay and Western blot
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)
Salient findings of our present study suggested that the 14‐day L‐NAME treatment elicited experimental hypertension, impaired cardiac contractile function and intracellular Ca2+ handing with little cardiac remodelling, in association with pronounced macrophage infiltration, inflammation, oxidative stress (O−2 production), loss of mitochondrial membrane potential and increased myocardial apoptosis
Summary
TUNEL staining was used to assess apoptosis in hearts from FVB and metallothionein transgenic mice with or without L‐NAME treatment. 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.
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