Objective: Mitochondrial dysfunction subsequent to increased oxidative-stress and alterations in energy metabolism is considered to play a role in the development of cardiac hypertrophy and its progression to failure, though the sequence of events remain to be elucidated. This study aimed at characterizing the impact of hypertrophy disease development on the activity and expression of mitochondrial NADP⁺-isocitrate dehydrogenase (mNADP⁺-ICDH), a metabolic enzyme that controls redox and energy status. We expanded on our previous finding of its inactivation through post-translational modification by the lipid peroxidation product 4-hydroxynonenal (HNE) in 7-week-old spontaneously hypertensive rat (SHR) hearts, before hypertrophy development (Benderdour et al. J Biol Chem 278: 45154, 2003). In the present study, we used 7-, 15-, and 30-week-old SHR and Sprague-Dawley (SD) rats with abdominal aortic coarctation. Results: Compared to age-matched control Wistar-Kyoto (WKY) rats, SHR hearts showed a significant 25% decrease of mNADP⁺-ICDH activity, which preceded in time (i) the decline in its protein and mRNA expression levels (between 10 and 35%), and (ii) the increase in hypertrophy markers. The chronic and persistent loss of mNADP⁺-ICDH activity in SHR was associated with enhanced tissue accumulation of HNE/mNADP⁺-ICDH and total HNE/protein adducts at all ages, and contrasted with the profile of changes in the activity of other mitochondrial enzymes involved in antioxidant or energy metabolism. Two-way ANOVA of the data revealed also a significant effect of age on most parameters measured in SHR and WKY rat hearts. The mNADP⁺-ICDH activity, protein and mRNA expression were reduced by 25 and 35% in coarctated SD rats, and were normalized by treatment of SHR or coarctated SD rats with renin-angiotensin system inhibitors, which prevented or attenuated hypertrophy. Conclusion: Our data show that cardiac mNADP⁺-ICDH activity and expression are differentially and sequentially affected in hypertrophy development and, to a lesser extent, with aging. Decreased cardiac mNADP⁺-ICDH activity, which is attributed at least in part to HNE adduct formation, appears to be a relevant early and persistent marker of mitochondrial oxidative stress-related alterations in hypertrophy development. Potentially, this could also contribute to the aetiology of cardiomyopathy.