Quantitative proteomic analysis reveals novel mitochondrial targets of estrogen deficiency in the aged female rat heart

Abstract

The incidence of myocardial infarction rises sharply at menopause, implicating a potential role for estrogen (E(2)) loss in age-related increases in ischemic injury. We aimed to identify quantitative changes to the cardiac mitochondrial proteome of aging females, based on the hypothesis that E(2) deficiency exacerbates age-dependent disruptions in mitochondrial proteins. Mitochondria isolated from left ventricles of adult (6 mo) and aged (24 mo) F344 ovary-intact or ovariectomized (OVX) rats were labeled with 8plex isobaric tags for relative and absolute quantification (iTRAQ; n = 5-6/group). Groups studied were adult, adult OVX, aged, and aged OVX. In vivo coronary artery ligation and in vitro mitochondrial respiration studies were also performed in a subset of rats. We identified 965 proteins across groups and significant directional changes in 67 proteins of aged and/or aged OVX; 32 proteins were unique to aged OVX. Notably, only six proteins were similarly altered in adult OVX (voltage-dependent ion channel 1, adenine nucleotide translocator 1, cytochrome c oxidase subunits VIIc and VIc, catalase, and myosin binding protein C). Proteins affected by aging were primarily related to cellular metabolism, oxidative stress, and cell death. The largest change occurred in monoamine oxidase-A (MAO-A), a source of oxidative stress. While acute MAO-A inhibition induced mild uncoupling in aged mitochondria, reductions in infarct size were not observed. Age-dependent alterations in mitochondrial signaling indicate a highly selective myocardial response to E(2) deficiency. The combined proteomic and functional approaches described here offer possibility of new protein targets for experimentation and therapeutic intervention in the aged female population.

Estrogen and the female heart

“…In contrast, increases were primarily observed for proteins involved in carbohydrate and amino acid metabolism (pyruvate dehydrogenase subunits) and enzymes of the tricarboxylic acid cycle. (143) Increased levels of HSP60 and mtHSP70 in aged OVX are consistent with previous studies in aged male hearts(144) and may be related to alterations in mitochondrial matrix protein import of nuclear-encoded enzymes, which may or may not be balanced by changes in proteolysis. Measurement of the activity and/or phosphorylation status(145) of these proteins is indicated for a more comprehensive characterization of metabolic alterations and substrate utilization in the aged female heart.…”

“…(143) Notably only 6 proteins were similarly altered in adult OVX (voltage-dependent ion channel 1, adenine nucleotide translocator 1, cytochrome c oxidase subunits VIIc and VIc, catalase, and myosin binding protein C), highlighting the specificity of the E 2 deficiency response in adult vs. aged female rats. Proteins affected by aging were primarily related to cellular metabolism, oxidative stress and cell death, with the largest change seen in monoamine oxidase-A, a potential source of oxidative stress.…”

“…About 50% of the identified proteins altered in aged OVX were associated with mitochondrial ATP production. (143) In aged, E 2 -deficient female hearts, reduced quantity of protein subunits of electron transport chain complex I (NADH dehydrogenase), II (succinate dehydrogenase), III (cytochrome bc 1 complex), IV (cytochrome c oxidase), and V (F 0 F 1 ATPase), and bidirectional changes in proteins involved in fatty acid substrate metabolism (acyl Co-A synthetase subunits) have been observed. In contrast, increases were primarily observed for proteins involved in carbohydrate and amino acid metabolism (pyruvate dehydrogenase subunits) and enzymes of the tricarboxylic acid cycle.…”

The Impact of Ovariectomy on Calcium Homeostasis and Myofilament Calcium Sensitivity in the Aging Mouse Heart

“…There is recent evidence that the production of reactive oxygen species (ROS) is increased in the aged OVX heart [51] and this may explain some of our findings. For example, increased ROS activity has been shown to increase ryanodine receptor activity, which could contribute to the increase in Ca 2+ sparks reported in our study [58].…”

“…Estrogen levels have been shown to decline with age in female rodent models [47] and this is exacerbated in the setting of OVX [50,51]. This suggests that the profound Ca 2+ dysregulation observed in the aging OVX heart is linked to low estrogen levels.…”