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The observation that hypoxia-induced K ATP channel activation is beneficial in mammals raises the possibility that a similar response in aquatic ectotherms could promote tolerance of environmental hypoxia. SarcK ATP channel currents have been recorded in isolated myocytes from the hearts of several teleost species ( Ganim et al., 1998 ; Paajanen and Vornanen, 2002 ), and there is evidence for the existence of cardiac mitoK ATP channels in fish ( MacCormack and Driedzic, 2002 ). In addition, nitric oxide synthase (NOS) has been localized in the hearts of goldfish ( Bruning et al., 1996 ), and NO has been shown to play a critical role, via cGMP, in regulating the heart and peripheral vasculature in a variety of teleost species ( Imbrogno et al., 2003 ; Jennings et al., 2004 ; Pellegrino et al., 2003 ). In goldfish, NO synthesized in cardiac myocytes plays a role in sarcK ATP channel activation during moderate hypoxia ( Lazamani Ankle boots petrol FjSUrrH
). Although it has been proposed that hypoxia-induced K ATP channel activation, whether in the heart or in the brain, contributes to the enhanced tolerance of environmental oxygen depletion exhibited by many ectothermic vertebrates ( Thom Browne Penny Loafer With Leather Sole In Pebble Grain Pg2YQR7C
; Pek-Scott and Lutz, 1998 ), this possibility has not been directly tested in fish.

The purpose of the present study, then, was to determine whether sarcK ATP or mitoK ATP channel activation exerts a cardioprotective effect during hypoxia in goldfish. This species is highly tolerant of depleted environmental oxygen; indeed, the congeneric crucian carp Carassius carassius retains normal cardiac function for 5 days of complete anoxia ( Stecyk et al., 2004 ). A cellular model of environmental hypoxia was used to examine the effects of altered channel activity on isolated myocytes. Our hypothesis was that the NO- and cGMP-dependent activation of cardiac K ATP channels would promote survival of isolated cells, thereby contributing to a suite of mechanisms that dramatically enhance tolerance of hypoxia in these fish. Preliminary results of this work have appeared in abstract form ( Zhu et al., 2004 ).

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Goldfish Carassius auratus L. were obtained from local suppliers and acclimated at 21°C in filtered, aerated 200 liter aquaria under a 12 h:12 h light:dark cycle. On the day of an experiment, goldfish of either sex were anesthetized by immersion in a solution of MS-222 (tricaine methanesulfonate, 0.2 g l –1 ; pH 7.6). The heart was rapidly excised and prepared for standard intracellular recording using the intact ventricle, or for enzymatic dispersion of individual myocytes.

Table 1. Sample size and assurance probabilities for observing criteria (i), (ii), and (iii) given = 0.025, = 0.1, (Δ , Δ ) = (3,0.45), ( , ) = (6,1), ( , ) = (0.5, 0.5), and = 0.1.

https://doi.org/10.1371/journal.pone.0180405.t001

Table 2. Sample size and assurance probabilities for observing criteria (i), (ii), and (iii) given = 0.025, = 0.1, (Δ , Δ ) = (3,0.45), ( , ) = (6,1), ( , ) = (0.5, 0.5), and = 0.3.

https://doi.org/10.1371/journal.pone.0180405.t002

Table 3. Sample size and assurance probabilities for observing criteria (i), (ii), and (iii) given = 0.025, = 0.1, (Δ , Δ ) = (3,0.45), ( , ) = (6,1), ( , ) = (0.5, 0.5), and = 0.5.

https://doi.org/10.1371/journal.pone.0180405.t003

Table 4. Sample size and assurance probabilities for observing criteria (i), (ii), and (iii) given = 0.025, = 0.1, (Δ , Δ ) = (3,0.45), ( , ) = (6,1), ( , ) = (0.5, 0.5), and = 0.7.

https://doi.org/10.1371/journal.pone.0180405.t004

In Tables 1 4 , we see the following phenomena. First of all, we found that as p 1 increases, the assurance probability of Criterion (i) increases. This is due to the fact that as p 1 increases, the observed overall results D k ’s will be increasingly dominated by the observed result from the first region, D 1 k ’s. Secondly, we have also observed that as p 1 increases, the assurance probability of Criterion (ii) increases first and then decrease later. This occurs because the observed result from regions other than the first region, ‘s, is gradually dominated by D 1 k ’s at first and is then completely dominated by D 1 k ’s later as p 1 increases. Also, the assurance probability of Criterion (iii) increases when p 1 increases, since the h i ’s decrease as p 1 increases.

Another feature we observed is that AP 1 > AP 2 in Tables 1 , 2 , 3 and 4 , given p 1 and γ k . This is due to the fact that since

Like Ikeda and Bretz [ 23 ] suggested, for Criterion (iii), it may be able to link the choice of ϕ to ( γ 1 , γ 2 ) = (0.5, 0.5) in order to ensure the same level of strictness of Criterion (i). For example, in Table 1 , setting ϕ = 0.17 in Criterion (iii) would closely ensure a similar level of strictness as Criterion (i) with p 1 = 0.4. Another point we wish to make is that the assurance probabilities of all criteria increase as ρ 12 increases. This makes intuitive sense because these two co-primary endpoints look more alike.

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