Prion-induced amyloid heart disease with high blood infectivity in transgenic mice

Abstract

We investigated extraneural manifestations in scrapie-infected transgenic mice expressing prion protein lacking the glycophosphatydylinositol membrane anchor. In the brain, blood, and heart, both abnormal protease-resistant prion protein (PrPres) and prion infectivity were readily detected by immunoblot and by inoculation into nontransgenic recipients. The titer of infectious scrapie in blood plasma exceeded 10(7) 50% infectious doses per milliliter. The hearts of these transgenic mice contained PrPres-positive amyloid deposits that led to myocardial stiffness and cardiac disease.

Fig. 1

Detection of PrPres in blood and extraneural tissues of tg mice infected with RML scrapie. (A) PrPres lining blood vessels within the corpus callosum of a tg mouse at 500 dpi with scrapie strain RML (PrPres staining in red). (B to E) PrPres was detected on immunoblots with monoclonal antibody D13. Similar results were observed with R30 antibody. (B) Immunoblot showing PrPres expression in blood cellular (C) and plasma (P) fractions following scrapie infection. For comparison, a sample (pos) equivalent to 10 mg of brain tissue isolated from the brain of a clinically sick RML scrapie-infected C57BL/6 (wt) mouse is shown. (C) Immunoblot showing 250 to 400 μl of plasma from six RML-infected tg mice at times ranging from 160 to 500 dpi. (D) Immunoblot analysis of PrPres in 20 mg of spleen, thymus, and heart from a tg mouse infected for 420 days. Similar results were observed with 10 additional mice. (E) Immunoblot analysis of heart homogenates from individual tg mice at various times after scrapie inoculation. Equivalent results were observed in at least three mice per time point. (F) Quantification of PrPres in blood cellular (C) and plasma (P) fractions relative to the amount of PrPres found in the brains of clinically sick WT mice after scrapie infection. Four times as much PrPres was identified in the plasma fraction as in the cellular fraction (*P ≤ 0.05). (G) Quantification of PrPres in the hearts of scrapie-infected tg mice relative to the amount of PrPres in the brains of clinically sick scrapie-infected WT mice. (♦) Each diamond represents data from a single mouse.

Fig. 2

Detection of PrPres deposits in cardiac samples from scrapie-infected tg mice using PrP monoclonal antibody D13. (A) Absence of PrPres in heart tissue in a scrapie-infected clinically ill WT mouse at 160 dpi. Similar results were obtained from an additional 10 scrapie-infected WT mice. (B) PrPres (red staining) in heart tissue at 400 days after RML scrapie infection of tg mice. PrPres deposition was observed primarily between myocytes and often around capillaries. (C and D) Adjacent sections from two mice at 450 dpi with RML scrapie were evaluated for PrPres (red) and amyloid (Thio-flavin S; green). PrPres most often localized with amyloid. A similar association between PrPres and amyloid was seen in more than 20 individual hearts from RML scrapie-infected tg mice. (E) Detection of widespread deposition of PrPres (pink) and (F) amyloid (Thioflavin S; green) from adjacent sections of heart from an infected tg mouse at 500 dpi. (G) PrPres could be found in or around myocytes at 500 dpi or later. (H) Detection of PrPres between myocardial myocytes and around blood-vessel endothelia in the heart of a mouse infected with 22L scrapie 715 days earlier.

Fig. 3

Hemodynamic assessment of RML scrapie-infected tg mice. Systolic and diastolic function was determined in tg mice with (red, n = 4) or without (black, n = 6) infection (400 to 500 days old or dpi) by pressure-volume analysis. Measurements are shown for a sequence of beats associated with the decrease in preload induced by transient occlusion of the inferior vena cava [left panel of (A) and (B)]. (A) Baseline hemodynamics. Emax, a measure of load-independent systolic function, is defined as the slope of the linear fit line of the end-systolic pressure-volume relation (black and red lines in the left panel). Average baseline indexes for Emax, end-diastolic volume (EDV), and end-diastolic pressure (EDP) are shown. (B) Measurement of passive cardiac relaxation (stiffness) after volume load. Cardiac stiffness was further assessed by infusing 0.5 ml of normal saline into the mouse as a volume load. After volume loading, the preload was altered by transient occlusion of the inferior vena cava. The slope of the linear fit of the end-diastolic pressure-volume relation (EDPVR) (black and red lines in the left panel) is proportional to the diastolic stiffness of the left ventricle. Means ± SE for the slope of the EDPVR, EDV, and EDP are shown. *P ≤ 0.05 as compared to uninfected tg mice.