ABSTRACT BACKGROUND Dysregulated proteostasis leading to accumulation of misfolded proteins, electron-dense aggregates (lipofuscin, LF), desmin preamyloid oligomers (PAOs), and proteotoxic stress is a hallmark of aging. We investigated how efficiently proteostatic adaptations to chronic cardiac cyclic adenosine monophosphate (cAMP)-dependent stress change with aging, in mice harboring a marked, cardiac-specific over-expression of adenylyl cyclase VIII (TG AC8 ). METHODS Protein quality control (PQC) mechanisms (ubiquitin proteasome system (UPS) and autophagy), including mitophagy, in TG AC8 and wild time littermates (WT) were evaluated in left ventricle (LV) at 3-4 months and at 17-21 months of age. Autophagy flux was evaluated in response to the lysosomotropic agent chloroquine (CQ), and protein synthesis was assessed according to the SUnSET puromycin method. RESULTS At 3-4 months of age, established autophagy markers such as the microtubule-associated protein 1 light chain 3 (LC3), p62, and their phospho-forms in TG AC8 LV were modulated to result in a more efficient autophagy. Flux was increased compared to WT. Lysosome performance (cathepsin L1 activity) in TG AC8 was also significantly increased, confirming higher cargo degradation and a more efficient proteostasis at 3-4 months. In advanced age, however, insufficiency of PQC mechanisms, manifested by a reduced proteasome activity, and an unbalanced autophagic flux, accelerated for markers such as LC3A and p62 within the context of a slower overall flux, led to an increase in the accumulation of aggresomes (increased ratio of insoluble/soluble protein fractions). In addition, although at 3-4 months canonical mitophagy signaling was increased (receptors PARKIN, p62-S403 and p62-S349 were upregulated) and non-canonical mitophagy signaling not engaged (receptor FKBP8 was decreased), both were upregulated at 18-21 months of age in TG AC8 , as protein levels of all receptors remained elevated. In old TG AC8 , the markedly impaired mitophagy, accompanied by the accumulation of lipofuscin (LF) bodies of aberrant sizes and brownish-to black pigment, increased concentration of cardiac preamyloid oligomers (PAOs) and elevated levels of cleaved desmin, tagged for ubiquitination, were characteristics of the age-effect compared to young TG AC8 . The rate of protein synthesis and levels of soluble aggregates were decreased, consequences of “normal” aging. CONCLUSIONS Proteostasis maintains cardiac health in TG AC8 in youth (3-4 months), but a long-term exposure to chronic pathophysiologic states that increase cardiac ubiquitination and stress, in response to sustained activation of the AC/cAMP/PKA/Ca 2+ axis, results in severely dysregulated proteostasis in TG AC8 vs WT, and is associated with proteostatic insufficiency, and accelerated cardiac aging. GRAPHIC ABSTRACT A graphic abstract is available for this article. GRAPHIC ABSTRACT/DIAGRAM Table. Proteostasis in young and aged TG AC8 in the context of cardiac stress in response to cardiac-specific constitutive stimulation of the AC/cAMP/PKA/Ca 2+ axis by α-MHC-driven AC8 overexpression.

Age-associated failure of efficient cardiac proteostatic adaptations to chronic cAMP-stress is associated with accelerated heart aging