Cardiac K(ATP) channel alterations associated with acclimation to hypoxia in goldfish (Carassius auratus L.).

Goldfish (Carassius auratus L.) are highly tolerant of environmental hypoxia, and with appropriate acclimation may survive and remain active for several days in the complete absence of oxygen. Previous work suggests that the hypoxia-induced activation of cardiac ATP-sensitive potassium (KATP) channels serves to increase tolerance of low oxygen in many species. For goldfish, we have previously characterized a nitric oxide (NO)- and cGMP-dependent pathway by which this channel activation occurs in acute hypoxia. The purpose of the present study was to resolve alterations in KATP channel activity and relevant gene expression in response to acclimation under moderately hypoxic conditions (2.6mg O2/L for seven days at 22°C). Intracellular action potential duration in excised ventricles from hypoxia-acclimated animals was significantly (p<0.05) reduced at both 50% and 90% of full repolarization relative to those from normoxia-acclimated fish. In cell-attached ventricular membrane patches from hypoxia-acclimated goldfish, sarcolemmal KATP channel open probability (NPo) was significantly enhanced vs. control. Of the two genes coding for the pore-forming subunits of cardiac KATP channels (Kir6.1 and Kir6.2), mRNA transcription of kcnj8 (revealed by quantitative real-time PCR) was unchanged while kcnj11 was downregulated in response to chronic low oxygen. The mRNA levels for hif1a (hypoxia inducible factor 1α) in the hearts of hypoxia-acclimated fish were significantly enhanced, as was nitric oxide synthase (nos2) and the sulfonylurea receptor regulatory subunit (sur2, abcc9). These data suggest that prior whole-animal acclimation to chronic hypoxia enhances cardioprotective sarcolemmal KATP currents by altering transcription of regulatory proteins.

Cardioprotective effects of K ATP channel activation during hypoxia in goldfish Carassius auratus.

The activation of ATP-sensitive potassium (K ATP) ion channels in the heart is thought to exert a cardioprotective effect under low oxygen conditions, possibly enhancing tolerance of environmental hypoxia in aquatic vertebrates. The purpose of this study was to examine the possibility that hypoxia-induced activation of cardiac K ATP channels, whether in the sarcolemma (sarcK ATP) or mitochondria (mitoK ATP), enhances viability in cardiac muscle cells from a species highly tolerant of low oxygen environments, the goldfish Carassius auratus. During moderate hypoxia (6-7 kPa), the activation of sarcK ATP channels was indicated by a reduction in transmembrane action potential duration (APD). This response to hypoxia was mimicked by the NO-donor SNAP (100 micromol l(-1)) and the stable cGMP analog 8-Br-cGMP, but abolished by glibenclamide or l-NAME, an inhibitor of NO synthesis. The mitoK ATP channel opener diazoxide did not affect APD. Isolated ventricular muscle cells were then incubated under normoxic and hypoxic conditions. Cell viability was decreased in hypoxia; however, the negative effects of low oxygen were reduced during simultaneous exposure to SNAP, 8-Br-cGMP, and diazoxide. The cardioprotective effect of diazoxide, but not 8-Br-cGMP, was reduced by the mitoK ATP channel blocker 5-HD. These data suggest that hypoxia-induced activation of sarcK ATP or mitoK ATP channels could enhance tolerance of low-oxygen environments in this species, and that sarcK ATP activity is increased through a NO and cGMP-dependent pathway.

A role for nitric oxide in hypoxia-induced activation of cardiac KATP channels in goldfish (Carassius auratus).

Hypoxia-induced shortening of cardiac action potential duration (APD) has been attributed in mammalian hearts to the activation of ATP-sensitive potassium (KATP) channels. Since KATP channels are also present at high densities in the hearts of vertebrate ectotherms, speculation arises as to their function during periods of reduced environmental oxygen. The purpose of the present study was to determine whether nitric oxide (NO) plays a role in cardiac sarcolemmal KATP channel activation during hypoxia in a species with a high degree of tolerance to low oxygen environments: the goldfish (Carassius auratus). Conventional intracellular and patch-clamp recording techniques were used to record responses from excised ventricles or isolated ventricular myocytes and inside-out patches, respectively, from fish acclimated at 21 degrees C. During moderate, substrate-free hypoxia (6.1 +/- 0.2 kPa), ventricular APD was significantly shortened at 50% and 90% of full repolarization, a response that was reversible upon reoxygenation and blocked by the KATP channel antagonist BDM. Under normoxic conditions, APD was also reduced in the presence of the NO-donor SNAP (100 micromol l(-1)). In cell-attached membrane patches, sarcolemmal KATP channel activity was enhanced after 10 min hypoxia, an effect that was reduced or eliminated by simultaneous exposure to BDM, to the guanylate cyclase inhibitor ODQ or to the NO synthase inhibitor L-NAME. In cell-free patches, KATP channel activity was abolished by 2 mmol l(-1) ATP but increased by SNAP; the cGMP analog 8-Br-cGMP (200 micromol l(-1)) also enhanced activity, an effect that was eliminated by BDM. Our data indicate that NO synthesized in cardiac myocytes could enhance sarcolemmal KATP channel activation during moderate hypoxia in goldfish. This response may serve a cardioprotective role by helping to conserve ATP or by reducing intracellular Ca2+ accumulation.