Fish plumbing is contrary. As the heart is the last organ that blood passes through before it returns to the gills, and with little direct blood supply to the ceaselessly contracting muscle, there are occasions when it could be on the verge of failure. ‘We know this can happen under certain conditions like exhaustive exercise in combination with hypoxia or elevated water temperature’, says Sarah Alderman from the University of Guelph, Canada. Added to the challenge of keeping the heart supplied with oxygen, Alderman explains that the haemoglobin that carries oxygen in fish blood is finely tuned to blood pH: the more acidic the red blood cells, the less able haemoglobin is to carry oxygen, which could prevent the red blood cells of exercising fish from picking up oxygen at the gills if they didn't have an effective pump to remove acid from the cells and restore the pH balance.
But Alderman and her colleagues, Till Harter, Tony Farrell and Colin Brauner from the University of British Columbia, Canada, also knew that fish can take advantage of a sudden drop in red blood cell pH to release oxygen rapidly at tissues – such as red muscle and the retina – when required urgently. An enzyme called carbonic anhydrase – which combines CO2 and water to produce bicarbonate and acidic protons, and vice versa – lies at the heart of this mechanism. Normally there is no carbonic anhydrase in blood plasma; however, the enzyme has been found in salmon red muscle capillaries, where it facilitates the reaction of protons – that have been extruded from the red blood cell – with bicarbonate to produce CO2, which then diffuses back into the red blood cell. The CO2 is then converted back into bicarbonate and protons in the blood cell, causing the pH to plummet and release a burst of O2 from the haemoglobin. Could salmon take advantage of this mechanism to boost oxygen supplies to the heart when the animals are working full out? Possibly, but only if carbonic anhydrase was accessible to blood passing through the heart.
Alderman and Jonathan Wilson began searching for the enzyme in salmon hearts. Using cobalt sulphide to stain sites where the enzyme accumulated, Alderman could clearly see the enzyme on the surface of the heart chambers. She also identified the gene that was expressed to produce the protein. Then, to be sure that carbonic anhydrase could contribute to dumping residual oxygen out of the blood in the heart, Alderman and Harter had to figure out a way of directly measuring pH inside the heart chambers to see whether the enzyme altered it. This type of experiment is usually performed using ground-up tissue in a test tube, but that wouldn't confirm that carbonic anhydrase in the heart chamber walls could alter blood pH, ‘So we decided to modify the assay and use the heart itself as the reaction vessel’, Alderman recalls.
Working closely together, the duo painstakingly developed a technique where they could measure the pH in the beating heart with pH probes that were thinner than a human hair. Eventually, the duo's persistence paid off and the pH in the heart plummeted as they fed CO2 into the pulsating chambers. And when they added a carbonic anhydrase inhibitor (produced by Claudia Supuran) to the fluid, the pH fall slowed dramatically. Carbonic anhydrase was responsible for the drop in pH.
‘The take-home message for our study is that the salmonid heart has an ace up its sleeve to help it overcome adverse conditions’, says Alderman, who is now eager to find out whether the fish play this card during their marathon upstream migration to spawn.