Age- and Hypertension-Associated Protein Aggregates in Mouse Heart Have Similar Proteomic Profiles

Abstract

Neurodegenerative diseases are largely defined by protein aggregates in affected tissues. Aggregates contain some shared components as well as proteins thought to be specific for each disease. Aggregation has not previously been reported in the normal, aging heart or the hypertensive heart. Detergent-insoluble protein aggregates were isolated from mouse heart and characterized on 2-dimensional gels. Their levels increased markedly and significantly with aging and after sustained angiotensin II-induced hypertension. Of the aggregate components identified by high-resolution proteomics, half changed in abundance with age (392/787) or with sustained hypertension (459/824), whereas 30% (273/901) changed concordantly in both, each P<0.05. One fifth of these proteins were previously associated with age-progressive neurodegenerative or cardiovascular diseases, or both (eg, ApoE, ApoJ, ApoAIV, clusterin, complement C3, and others involved in stress-response and protein-homeostasis pathways). Because fibrosis is a characteristic of both aged and hypertensive hearts, we posited that aging of fibroblasts may contribute to the aggregates observed in cardiac tissue. Indeed, as cardiac myofibroblasts "senesced" (approached their replicative limit) in vitro, they accrued aggregates with many of the same constituent proteins observed in vivo during natural aging or sustained hypertension. In summary, we have shown for the first time that compact (detergent-insoluble) protein aggregates accumulate during natural aging, chronic hypertension, and in vitro myofibroblast senescence, sharing many common proteins. Thus, aggregates that arise from disparate causes (aging, hypertension, and replicative senescence) may have common underlying mechanisms of accrual.

Keywords: aging (cardiac); cardiovascular diseases; hypertension; neurodegenerative diseases; protein aggregates.

Fig. 1

Aggregated proteins increase in cardiac cells with natural aging, hypertension, and in vitro senescence. Aggregates were isolated by differential centrifugation and sarcosyl-insolubility. Panels A, B, D, E, G and H show typical separations of proteins from hearts of young mice (A, 3.5 months old) or aged mice (B, 30 months), with data summarized in panel C; saline-infused normotensive young mice (D), angiotensin-II infused hypertensive young mice (E), data summarized in F. Aggregate proteins from mouse cardiac myofibroblasts at 3 MPD (early-passage, G) and 15 MPD (”senescent”, H), with combined data summarized in panel I. Histograms (panels C, F and I) present data as mean ± SD, for ImageJ quantitation of aggregate-protein signal from 3 different mice or in-vitro cultures.

Fig. 2

Venn diagrams indicate the overlap of identified proteins that increased (A) or decreased (B) significantly in abundance within cardiac aggregates, as mice aged naturally, or were made hypertensive by angiotensin-II infusion, or as cardiac myofibroblasts underwent replicative aging in vitro. All proteins included in these totals differed with at least nominal significance (chi-squared P<0.05) for the indicated comparisons.

Fig. 3

Representative western blots for HSP90, fibronectin, vimentin, 14-3-3, ApoE and Clusterin from young, aged and hypertensive mice. Multiple isoforms (e.g. for HSP90, 14-3-3, ApoE) are not distinguished. ImageJ quantitations of each protein are indicated by bars and the spectral counts identified by mass spectrometry are indicated by numbers above each bar. For each gel lane with maximum intensity is used as 100 percent. The significance is indicated by *, **= ≥0.01 and *= ≥0.05. Molecular weight of each protein HSP90 =90kD, Fibronectin -220kD, Cytochrome C - 15kD, 14-3-3 = 29kD, Apo E - 44kD and clusterin - 36kD.