Mechanism of age-related cardiac dysfunction: Role of iNOS

Abstract

It is well documented that in the human free of pathological change, there is cardiac diastolic dysfunction with decreased reserved systolic function. Inducible nitric oxide synthase (iNOS) expressed in cardiac myocytes is related to heart failure of various etiologies. We hypothesized that the high levels of nitric oxide (NO) produced by iNOS over-expressed in the myocardium caused age-related cardiac dysfunction by affecting calcium cycling proteins. To test our hypothesis, first, we validated the volumetric measurement of the conductance catheter system (CCS) in mice. The heart function characterized by CCS decreased in aged mice (16-month-old, n = 9) compared to Young mice (6-month-old, n = 9). The left ventricular (LV) dysfunction in aged WT mice was reversed by iNOS specific inhibitors, aminoguanidine (AMG, 10 mg/Kg, i.v. or infusion, n = 15) and S-methyl-isothiourea (MITU, 3 mg/Kg, i.v. n = 7) separately and worsened by substrate L-arginine (10 mg/Kg, i.v. n = 7). All three drugs had no effects on young WT mice or old (16-month-old) iNOS knockout (KO) mice. Cardiac iNOS mRNA and protein expression were identified with northern blot, western blot and immunohistochemistry singularly in old WT mice and not in young WT mice. The NOx and cGMP levels were significantly higher only in the old WT mice (n = 6) compared to young WT mice and cGMP levels decreased to normal with AMG administration in the old WT mice. Northern blot showed the expression of ryanodine receptor, sarco/endoplasmic reticulum (SERCA 2a) did not change in the aging process. However western blots showed that total phospholamban (PLB), phosphorylated PLB (p < 0.05) and more importantly the ratio of phosphorylated PLB to total PLB significantly decreased in aged mice (p < 0.01). AMG (10 mg/kg bolus iv) significantly improved diastolic function only in the old WT mice with decreased tau and increased dP/dtmin, but had no effects on both young or old PLB knockout mice. Taken together, we concluded that iNOS overexpression was one of the mechanisms leading to age-related cardiac dysfunction by affecting phosphorylation of PLB via iNOS/NO/cGMP pathways and inhibition of iNOS could reverse the age-related cardiac dysfunction.