John R. Bailey

CARDIAC ADAPTATIONS TO LOW TEMPERATURE IN NON-POLAR TELEOST FISH

Acclimation of teleost fish to low temperature results in a number of well-characterized adaptive responses in skeletal muscle. Our understanding of the response of cardiac muscle to such a challenge though is still poorly understood. It is considered of interest to learn more about the impact of chronic low temperature exposure on the cardiac response since the heart is required to support other tissues through the delivery of oxygen and metabolic fuels and this system may be a useful model in which to identify loci of cardiac plasticity. An overview of the available data reveals a number of fundamental responses. These include an increase in heart mass, an increase in maximal contraction frequency, an increase in complex lipid biosynthesis, and an increase in aerobic-based fatty acid metabolism. Two different strategies seem to emerge and lead to working hypotheses. As one example, rainbow trout develop larger hearts which may lead to enhanced stroke volume. The net accretion of protein is associated with either a more effective use of the available protein synthetic machinery and/or a decrease in protein degradation. The net accretion of lipid is associated with an increased rate of biosynthesis. Energy metabolism at low temperature appears to be much more dependent upon the oxidation of fatty acids. As another example, perch acclimated to low temperature show enhanced maximal rates of contraction which may be associated with increase in the volume of sarcoplasmic reticulum. The metabolic processes necessary to supply ATP appear quite adequate and require no further expansion as evidenced by constant mitochondrial volume density and activity of mitochondrial marker enzymes. The data however, in both cases are fragmentary and call for comprehensive studies on selected species.

OXYGEN UPTAKE BY ISOLATED PERFUSED FISH HEARTS WITH DIFFERING MYOGLOBIN CONCENTRATIONS UNDER HYPOXIC CONDITIONS

Hearts from three species of fish with varying myoglobin content were perfused with stepwise changes in input perfusate PO2 from approximately 160 to 10 mmHg. Flow through the heart, rate of contraction, and afterload were kept constant. This standardized stroke volume and bulk flow of perfusate to the myocytes since these hearts are nourished by the fluid in the ventricular lumen. In some cases NaNO2 was added to the perfusion medium to decrease existing levels of functional myoglobin. Myoglobin-rich hearts were able to extract a constant amount of oxygen until perfusate PO2 had fallen below 80 mmHg. At this point oxygen uptake began to decline. These hearts consumed oxygen until input PO2 was 10 mmHg or less. When normoxic conditions were restored the myoglobin-rich hearts showed complete recovery. Performance was maintained at a constant level over the entire range of input PO2. Myoglobin-poor hearts and nitrite-treated hearts were unable to sustain constant levels of oxygen consumption in the face of a declining perfusate PO2. These hearts were unable to extract oxygen from the medium and failed at perfusate PO2s of 40 mmHg for naturally myoglobin-poor hearts and 30 mmHg for nitrite-treated hearts. Half-maximal oxygen consumptions were attained by myoglobin-rich hearts at lower input PO2s than either myoglobin-poor or nitrite-treated hearts. The impact of myoglobin in intact heart is apparent at relatively high extracellular PO2s (40-80 mmHg) in this model system.

Impact of an Acute Temperature Change on Performance and Metabolism of Pickerel (Esox niger) and Eel (Anguilla rostrata) Hearts

Mechanical performance, oxygen consumption (Ṁo2), and the maximal activities of key enzymes of energy metabolism were assessed in chain pickerel (Esox niger) and American eel (Anguilla rostrata) hearts over a temperature range normally experienced by these fish. Ventricle strips from pickerel could be paced up to 48 contractions min−1 at both 5° and 15°C. Eel ventricle strips responded at 48 contractions min−1 at 15° C but at 5° C could not be paced faster than 18 contractions min−1. At low temperatures, time to 50% relaxation was especially prolonged in eel ventricle strips, and increases in extracellular calcium resulted in large increases in resting tension. These findings suggest that calcium extrusion from the myocyte is impaired. Maximum output of perfused, isolated pickerel hearts occurred at frequencies of 30 and 36 contractions min−1 at 5° and 15°C. Values for eel hearts were 18 and 30 contractions min−1. Pickerel hearts had the same power output at both temperatures. Power output of eel hearts was lower at 5° than 15°C. Oxygen consumption of perfused hearts displayed a Q10 of about 1.9 for both pickerel and eel. Maximal in vitro activities of hexokinase, carnitine acyl-CoA transferase, citrate synthase, and ATPase were determined at 5° and 15° C. Comparison of Ṁo2 values with enzyme activities reveals that the catalytic potential to provide ATP is in excess of resting energy demand at both temperatures. The impairment of eel heart performance at low temperature cannot be attributed to an inability to metabolize glucose or fatty acids. The findings with isolated preparations are consistent With maintenance of activity by pickerel and presumptive in situ cardiac function over a wide temperature range and with the lack of activity displayed by eel at low temperature in association with known reduced cardiac performance.

HEART GROWTH ASSOCIATED WITH SEXUAL MATURITY IN MALE RAINBOW TROUT (ONCORHYNCHUS MYKISS) IS HYPERPLASTIC

Abstract Ventricle mass increases, relative to body mass, with sexual maturity in male rainbow trout. It is not known whether this increase is due to hyperplasia or hypertrophy. We examined this question in hatchery-reared male and female rainbow trout over a 16-week study. Fork length, body, heart (ventricle), and gonad mass were recorded. Ventricles were analyzed for DNA, RNA, and protein content. We confirm that ventricle growth occurs in males but not in females. There was a slight but significant decrease in protein content of hearts from male trout with increasing heartsomatic index but protein content did not change in hearts from female trout. There was no change in RNA/protein ratio in either male or female trout hearts indicating that increased protein synthesis in male trout hearts was due to an increase in RNA activity. The DNA/protein ratio showed a significant increase in male trout hearts, but no change in female trout hearts. We conclude that the major part of the increase in ventricle mass in male trout hearts is due to hyperplasia.

Impact of acute temperature transition on enzyme activity levels, oxygen consumption, and exogenous fuel utilization in sea raven (Hemitripterus americanus) hearts

SummaryThe impact of an acute temperature transition between 5 °C and 15 °C on energy metabolism and performance of sea raven (Hemitripterus americanus) heart was assessed. Maximal in vitro activity of hexokinase was 1.2 and 3.7 μmol · min-1 · g-1 at 5 °C and 15 °C, respectively. Carnitine palmitoyl transferase and carnitine palmitoleoyl transferase activities were 0.07 μmol · min-1 · g-1 at 15 °C and declined substantially at 5 °C. Oxygen consumption and power output of perfused isolated hearts offered glucose alone as a metabolic fuel decreased significantly between 15 °C and 5 °C. When palmitoleate was included in the perfusion medium, oxygen consumption and power development remained constant between 15 °C and 5 °C, suggesting that glucose alone was not an adequate metabolic fuel at low temperature. However, maximal in vitro activity of HK implied that the catalytic potential at this locus was quite capable of meeting demands of carbon flow, while the maximal in vitro activity of the carnitine acyl CoA transferases implied that fatty acid metabolism should be greatly compromised at low temperatures. In an effort to resolve the contradiction, hearts were perfused with medium containing 14C-glucose or 14C-palmitate. Rates of 14CO2 production from labelled metabolic fuels could account for only about 2% of the oxygen consumption rates. Most of the label from 14C-glucose was incorporated into the glycogen and lipid fractions and label from 14C-palmitate was incorporated into the lipid fraction. The net incorporation rates of label into intracellular pools were temperature insensitive over the range 5–15 °C. The incorporation of exogenous glucose into the lipid fraction suggests that activity of the entire glycolytic pathway was maintained over the temperature range. Thus, the relatively low rate of oxygen consumption of hearts perfused with glucose alone as an exogenous substrate cannot be attributed to a limitation of glucose catabolism. The alternative explanation is that the presence of fatty acids induces an increase in oxygen consumption, especially at 5 °C. It is speculated that this is due to alterations in Ca2+ balance.

Anoxic performance of the American eel (Anguilla rostrata L.) heart requires extracellular glucose

The importance of extracellular glucose in the maintenance of performance of the heart of the American eel (Anguilla rostrata Le Sueur (L.) Under anoxia was assessed under a variety of experimental conditions. Ventricular strips, electrically paced at 36 bpm, in N(2)-gassed medium maintained the imposed pace rate and generated approximately 25% of the initial twitch force of contraction for at least 60 min when glucose was present in the medium. But ventricular strips challenged without glucose in the medium failed to maintain the pacing rate within 5-10 min. Isolated and intact, perfused hearts maintained pressure and followed an imposed pace rate of 24 bpm for at least 2 hr, under anoxic conditions, if glucose was present in the medium. But without glucose in the medium isolated hearts failed within 30 min. Endogenous glycogen stores were utilized in hearts perfused with medium containing NaCN to impair oxidative phosphorylation. The presence of glucose in the medium did not protect against glycogen mobilization. The data indicate that exogenous glucose is necessary to maintain performance under anoxia at high workloads and physiological Ca(2+) levels. Finally, ventricular strips treated with NaCN and forced to contract at 24 bpm lost 70% of initial twitch force. Increasing extracellular Ca(2+) concentration stepwise from 1.5 to 9.5 mM restored twitch force to approximately 50% of the initial level and this response was not dependent on exogenous glucose. However, glucose was required to maintain resting tension even under normoxic conditions in the face of a Ca(2+) challenge.

Maintenance of resting tension in the American eel (Anguilla rostrata L.) heart is dependent upon exogenous fuel and the sarcoplasmic reticulum

The relationship between extracellular glucose and management of cell Ca(2+) in the heart of the American eel (Anguilla rostrata) was indirectly assessed by monitoring the performance of isolated ventricular strips at 20 degrees C. Twitch force increased in ventricular strips under specific conditions of 30 bpm pacing and an extracellular Ca(2+) challenge from 1.5 to 9.5 mM. The response was independent of any exogenous metabolic fuel in the medium. Resting tension was maintained when glucose was available, but in the absence of a metabolic fuel, resting tension increased in response to the increase in extracellular Ca(2+) level. When ventricular strips were treated with iodoacetate to inhibit glycolysis, a Ca(2+) challenge resulted in a decrease in twitch force in association with an approximately equivalent increase in resting tension even in the presence of exogenous glucose. However, when pyruvate (5 mM) was substituted as a metabolic fuel, twitch force increased as a function of extracellular Ca(2+), and resting tension was maintained in the presence of iodoacetate. Therefore, there is a need for an extracellular fuel but not a specific metabolic requirement for glucose to maintain the performance characteristics, which are presumably related to the management of intracellular Ca(2+) levels. Ventricular strips were treated with ryanodine to inhibit Ca(2+) release and uptake by the sarcoplasmic reticulum (SR). Ryanodine treatment impaired postrest potentiation at high extracellular Ca(2+) levels. In the presence of ryanodine, the protective effect of glucose on the increase in resting tension in the face of an extracellular Ca(2+) challenge was eliminated. Considered together, the results reveal that the heart of the American eel has a requirement for an extracellular fuel to manage intracellular Ca(2+) at high Ca(2+) loads, and that the SR plays a role in the beat-to-beat regulation of Ca(2+) at a frequency of 30 bpm, high Ca(2+) load, and 20 degrees C.

Oxygen consumption in myoglobin-rich and myoglobin-poor isolated fish cardiomyocytes.

The function of myoglobin at the cellular level was investigated by comparing O2 consumption in isolated myoglobin-rich cardiac myocytes from the sea raven (Hemitripterus americanus) and myoglobin-poor myocytes from the ocean pout (Macrozoarces americanus). O2 consumption by sea raven myocytes, 0.21 +/- 0.04 microM O2/10(6) cells.min-1, was significantly higher than O2 consumption by ocean pout myocytes, 0.10 +/- 0.07 microM O2/10(6) cells.min-1 at high PO2. O2 consumption in sea raven myocytes treated with sodium nitrite was not significantly different than that in untreated myocytes at high PO2, but it was significantly lower than controls at low PO2. O2 consumption of sea raven myocytes treated with the mitochondrial uncoupler CCCP was not significantly different from that of control myocytes at high PO2, but it was significantly greater than untreated controls at low PO2. In ocean pout preparations, O2 consumption by nitrite-treated myocytes was significantly higher than that of untreated myocytes at high PO2, but it was not different from that of controls at low PO2. CCCP-treated ocean pout myocytes had a significantly higher oxygen consumption than that of untreated myocytes at high PO2, but oxygen consumption was not different from that of controls at low PO2. The CCCP-activated O2 consumption at low PO2 was myoglobin-dependent in that CCCP alone resulted in a threefold increase in sea raven cells over controls but had no impact on sea raven cells in the presence of nitrite or ocean pout cells treated with CCCP alone. This study further supports the contention that myoglobin only plays an important role in oxygen metabolism at low extracellular PO2s.

Effects of Acute Temperature Change on Cardiac Performance and Oxygen Consumption of a Marine Fish, the Sea Raven (Hemitripterus americanus)

Sea ravens (Hemitripterus americanus), subjected to a temperature transition from 10° to 5° C over a 3-h period, showed a decrease in ventral aortic pressure from 42 to 18 cmH₂0 and a decrease in heart rate from 31 to 14 bpm. Isolated hearts, per-fused at 10° C with media containing 1 mM CaCl₂, were able to perform as well as in vivo hearts. When the in vitro preparation was taken from 10° to 5° C there was a small but significant decrease in performance and oxygen consumption over frequencies ranging from 12 to 48 bpm. The decrease in function in vivo was much more substantial than could be accounted for by the direct impact of temperature on the isolated heart. The small but significant decrease in performance by the isolated heart was not due to an impairment of energy-production mechanisms or limited extracellular calcium, as an increase in extracellular calcium resulted in an increase in oxygen consumption with no alteration in contractile performance.

A Newly Synthesised Molybdenum/Ascorbic Acid Complex Alleviates Some Effects of Cardiomyopathy in Streptozocin-Induced Diabetic Rats

AbstractBackground: Exogenous insulin does not prevent cardiac failure in patients with type 1 diabetes mellitus and a cardioprotective insulin mimic is greatly needed. Certain transition metals are known to act as insulin mimics and may be cardioprotective. In this study, the ability of a newly synthesised molybdenum/ascorbic acid complex to strengthen cardiac function was investigated. Methods and design: Male CD rats were assigned to one of five groups: non-diabetic control, non-diabetic control treated with molybdenum/ascorbic acid complex, diabetic treated with sodium ascorbate, diabetic treated with molybdenum/ ascorbic acid complex and untreated diabetics. Type 1 diabetes was induced by streptozocin injection. Once diabetes was confirmed, treatment was initiated by adding either the molybdenum/ascorbic acid complex or sodium ascorbate to the drinking water and continued for 6 weeks. Following the treatment period, the animals were terminated, and their hearts were excised and mounted in a working heart perfusion apparatus. Blood samples were taken for plasma glucose and plasma lipid level determination. Cardiac function was evaluated using 1 hour of low-flow ischaemic stress followed by 30 minutes of reperfusion. Results: Hearts from the animals treated with the molybdenum/ascorbic acid complex displayed the best aerobic performance of all the diabetic animals. Blood glucose levels and blood lipid levels were significantly lower in animals treated with the complex than in other diabetic animals. The group treated with the complex also had a lower drinking rate than the other diabetic groups. Furthermore, hearts from animals treated with the molybdenum/ascorbic acid complex showed a greater degree of recovery from low-flow ischaemia than any other group. Conclusions: The molybdenum/ascorbic acid complex showed some significant insulin-mimic and cardioprotective effects. Further development of this complex could provide a drug useful for alleviating some of the cardiovascular problems associated with diabetes mellitus.