Examining cardiac performance of tunas at the cellular level

Are all tunas alike? It is true that they are all swift, powerful swimmers that benefit from high metabolic rates – and that in order to support these rates, they have evolved into a state of high heart rates. Consider the skipjack tuna, which has been clocked at a heart rate of over 200 beats-per-minute. But is the cardiac stamina of the cold water (endothermic) tuna, such as the bluefin, albacore and yellowfin, the same as that of its warm water (ecothermic) sister the mackerel? Why should it matter?

A research team from Stanford University’s Tuna Research and Conservation Center has investigated the intrinsic differences among these tunas. Their findings suggest that a key step in the evolution of the tuna’s high heart and metabolic rates is the result of an increase a key protein –SERCA2. Their findings also suggest that high levels of the enzyme in the bluefish tuna’s heart may be important for its ability to retain its cardiac function at cold temperatures. While a better understanding of the tuna may seem of little consequence, what researchers learn about the adaptation of these magnificent fish helps us look beyond ourselves and to appreciate the lives of those living below sea level.

A New Study

The authors of the new study, entitled “Temperature Dependence of the Ca2+-ATPase (SERCA2) in the Ventricles of Tuna and Mackerel,” are Ana M. Landeira-Fernandez, Jeffery M. Morrissette, Jason M. Blank and Barbara A. Block, all of the Tuna Research and Conservation Center, Hopkins Marine Station, Stanford University, Pacific Grove, CA. Their findings appear in the Articles in Press section of the American Journal of Physiology – Regulatory, Integrative and Comparative Physiology, one of 14 scientific journals published monthly by the American Physiological Society (APS) (www.the-aps.org).

Methodology

In this study, the role of Ca2+ cycling by sarcoplasmic reticulum in the hearts of tunas and mackerels was investigated by comparing the properties of the Ca2+ATPase (SERCA2) in ventricular microsomal preparations. To conduct the experiment the research team followed the methodology noted below:

Fish:
Four Pacific bluefin tuna (Thunnus orientalis), four albacore tuna (Thunnus alalunga) and three yellowfin tuna (Thunnus albacares) were caught off the coast of California, euthanised and their hearts immediately removed. Ventricles were sliced into small pieces, free-clamped and the tissues stored for up to four months prior to the experiments. Twenty Pacific mackerel (Scomber japonicus) were also caught, transported to the Tuna Research and Conservation Center, euthanized and their hearts excised with the ventricles immediately freeze-clamped in liquid nitrogen.

Ventricular Sarcoplasmic Reticulum Vesicle Isolation:
Sarcoplasmic reticulum enriched microsomes were prepared. The protein concentration was determined according to Bradford.

Ca2+ Uptake:
For measurement of Ca2+ uptake, 0.6mg/ml (tunas) or 1 mg/ml (mackerel) of microsomes were added to a cuvette containing 50 mM Mops-Tris pH 7.0, 100 mM KCl, 1 mM MgCl2, 10 mM sodium azide, 10 mM potassium-oxalate, 5 mM creatine phosphate, 10 g/ml creatine kinase (as an ATP regenerating system) and 1.5 M Ca2+ sensitive fluorescent dye fura-2. The cuvette was placed in a spectro- fluorophotometer and the vesicles and media were allowed to equilibrate. Ca2+ uptake was stimulated by the addition of 1.5 mM MgATP. After the steady state was reached, CaCl2 was added to a final concentration of 10 M and the reaction was allowed to reach steady state again. At the end of each experiment, 1 l triton X-100 or 1.5 M of the Ca2+ ionophore A23187 was added in order to collapse the SR Ca2+ gradient. The initial rate of Ca2+ uptake was calculated from the t(1/2) (the time for one-half of the Ca2+ to be taken up into the vesicles).

ATPase Activity:
The ATP hydrolysis was measured. The Mg2+-dependent activity was measured in the presence of 2 mM EGTA. The Ca2+-ATPase activity was determined by subtracting the Mg2+-dependent activity from the total activity measured in the presence of both Mg2+ and 10 M Ca2+. At the desired temperature, the reaction was started by the addition of 0.1 mg/ml SR microsomes and stopped by the addition of 10 % TCA. Aliquots from the reaction medium were taken at times of 3, 5, 10, and 30 min.

SDS-PAGE and Western-Blot Analyses:
Microsomal preparations from bluefin, yellowfin, albacore and mackerel ventricles were separated by electrophoresis. Proteins were visualized with silver stain. For western blot analysis, proteins were transferred to PVDF membranes and probed with a polyclonal antibody specific to cardiac SERCA2. Blots were developed using a secondary antibody kit. SERCA2 content was assessed.

Results

The researchers found that:

• The measurements of oxalate-supported Ca2+ uptake in SR enriched ventricular vesicles indicated that tunas were capable of sustaining a rate of Ca2+-uptake that was significantly higher than the mackerel;
• among tunas, the cold tolerant bluefin had the highest rates of SR Ca2+ uptake and ATPase activity;
• the differences among Ca2+ uptake and ATP hydrolysis rates did not seem to result from intrinsic differences between the SERCA2 present in the different tunas, as shown by their similar temperature sensitivities and similar values for activation energy; and
• Western blots revealed that increased SERCA2 protein content is associated with the higher Ca2+ uptake and ATPase activities seen in bluefin ventricles compared to albacore, yellowfin and mackerel.

Conclusions

This study reveals that distinct cellular differences in the expression of a key protein –SERCA2 – are associated with the beat-to-beat contraction of tuna and mackerel hearts. It suggests that there are fundamental differences in the way these fish initiate myocyte contraction, and provides further evidence for the role of the SR Ca2+-ATPase in cardiac function of tunas.

The American Physiological Society (APS) was founded in 1887 to foster basic and applied science, much of it relating to human health. The Bethesda, MD-based Society has more than 10,000 members and publishes 3,800 articles in its 14 peer-reviewed journals every year.