Fig. 2. Experimental protocols used to examine the effect of duration of severe hypoxia (P_O =5–10 mmHg) on in situ trout heart function. Hearts were exposed to one of four treatment protocols: (A) control (oxygenated perfusion) (N=7), (B) 10 min (N=7), (C) 20 min (N=7) or (D) 30 min (N=8) of severe hypoxia. In each protocol, the solid line represents the end-diastolic pressure developed by the ventricle, determined by adjusting the height of the output pressure (P_OUT) head. P_OUT was set to either a physiologically realistic level of 50 cmH₂O, or a sub-physiological level of 10 cmH₂O. The arrows () mark the initial cardiac stretch, where input pressure (P_IN) was raised to elicit a cardiac output () of 30 ml min^–1 kg^–1. The steps identify the maximum cardiac output tests (_MAX1 and _MAX2), where P_IN was raised sequentially from 3 cmH₂O to 4 cmH₂O, and finally to 4.5 cmH₂O. The shaded rectangles indicate periods of induced severe hypoxia (P_O =5–10 mmHg). During hypoxia, P_IN was not adjusted and was allowed to fall. During all periods of oxygenated cardiac perfusion, was maintained at a physiologically resting level of 16–17 ml min^–1 kg^–1 by adjusting P_IN as needed. The small, open squares represent points at which perfusate samples (1 ml) were collected for biochemical analysis.