Interrelationship Between Contractility, Protein Synthesis and Metabolism in Mantle of Juvenile Cuttlefish (Sepia officinalis)

Abstract

Young juvenile cuttlefish (Sepia officinalis) can grow at rates as high as 12% body weight per day. How the metabolic demands of such a massive growth rate impacts muscle performance that competes for ATP is unknown. Here, we integrate aspects of contractility, protein synthesis, and energy metabolism in mantle of specimens weighing 1.1 g to lend insight into the processes. Isolated mantle muscle preparations were electrically stimulated and isometric force development monitored. Preparations were forced to contract at 3 Hz for 30 s to simulate a jetting event. We then measured oxygen consumption, glucose uptake and protein synthesis in the hour following the stimulation. Protein synthesis was inhibited with cycloheximide and glycolysis was inhibited with iodoacetic acid in a subset of samples. Inhibition of protein synthesis impaired contractility and decreased oxygen consumption. An intact protein synthesis is required to maintain contractility possibly due to rapidly turning over proteins. At least, 41% of whole animal ṀO₂ is used to support protein synthesis in mantle, while the cost of protein synthesis (50 μmol O₂ mg protein^-1) in mantle was in the range reported for other aquatic ectotherms. A single jetting challenge stimulated protein synthesis by approximately 25% (2.51-3.12% day^-1) over a 1 h post contractile period, a similar response to that which occurs in mammalian skeletal muscle. Aerobic metabolism was not supported by extracellular glucose leading to the contention that at this life stage either glycogen or amino acids are catabolized. Regardless, an intact glycolysis is required to support contractile performance and protein synthesis in resting muscle. It is proposed that glycolysis is needed to maintain intracellular ionic gradients. Intracellular glucose at approximately 3 mmol L^-1 was higher than the 1 mmol L^-1 glucose in the bathing medium suggesting an active glucose transport mechanism. Octopine did not accumulate during a single physiologically relevant jetting challenge; however, octopine accumulation increased following a stress that is sufficient to lower Arg-P and increase free arginine.

Keywords: anaerobic metabolism; cycloheximide; glucose; iodoacetic acid; jetting; octopine.

FIGURE 1

Schematic of experimental design. (A) Contractility study. (B) ṀO₂, protein synthesis, and metabolite levels.

FIGURE 2

Relative peak stress development by isometrically contracting mantle muscle strips from cuttlefish (Sepia officinalis). Preparations were initially stimulated to contract six times at 0.2 Hz. Stress developed during this period served as the level at which all further challenges were related. All experiments involved paired preparations. Bathing media contained glucose (open bars) plus additional 25 μM CHX (stippled bars; left panels) or additional 1 mM IAA (stippled bars; right panels). Panels (A,B) – Δ stress development following a 10 min rest followed by a second wave of six contractions at 0.2 Hz. Panels (C,D) – Δ stress development following contractions for 30 s at 30 Hz [immediately following (A,B)]. Panels (E,F) – Δ stress development following 1 h rest [following (C,D)]. ^∗Significant difference in response of matched preparation in media containing glucose alone relative to preparations in media with either additional CHX or IAA. N = 6 for CHX experiments and N = 5 for IAA experiments. Box and whisker plots show the range and median value.

FIGURE 3

Rate of oxygen consumption and protein synthesis by isolated mantle muscle strips from cuttlefish (Sepia officinalis). One of two paired mantle strips was stimulated to contract for 30 s at 3 Hz simulating jetting behavior. Oxygen consumption and protein synthesis was then measured during the recovery period. (A) Representative trace of rate of oxygen consumption. The curve with squares and solid line represents oxygen consumption by a mantle preparation that had been forced to contract; the dashed line with circles is the matched unstimulated paired preparation. (B) Average rate of oxygen consumed over a 40–60 min period following contraction. (C) Rate of protein synthesis measured over a 1 h period following contraction. Open bars represent unstimulated preparations; stippled bars indicate preparations that were stimulated. Text below x-axis shows additions to media. For CHX and IAA additions, preparations were incubated for 10 min prior to stimulation, during stimulation, and throughout the post contraction incubation period. For ṀO₂ measurements N = 7 for glucose; N = 4 for CHX and IAA. For protein synthesis N = 6 in all cases. Values with different letters are significantly different. ^∗Significant difference between unstimulated and stimulated preparations. Box and whisker plots show the range and median value.

FIGURE 4

Glucose, octopine, and arginine levels following 1 h recovery from intense contractility. One of two paired mantle muscle sheets from cuttlefish (Sepia officinalis) was stimulated to contract for 30 s at 3 Hz simulating jetting behavior. (A,C,E) tissue concentrations after a 1 h period following contraction. (B,D) rate of uptake or release of metabolite into the bathing medium during the 1 h period following contraction. Open bars represent unstimulated preparations; stippled bars indicate preparations that were stimulated. Text below x-axis shows additions to media. For CHX and IAA additions, preparations were incubated for 10 min prior to stimulation, during stimulation, and throughout the post contraction incubation period. N = 6 under all conditions except tissue glucose in stimulated preparations incubated with CHX, tissue octopine in unstimulated preparations incubated with CHX, and media glucose in unstimulated preparations incubated with IAA where N = 5. Values with different letters are significantly different. ^∗Significant difference from zero (one sample t-test) for rate of glucose appearance in bathing medium. Box and whisker plots show the range and median value.

FIGURE 5

Glucose (A), octopine (B), and arginine (C) levels in mantle muscle sheets immediately following intense contractility. One of two paired mantle sheets from cuttlefish (Sepia officinalis) was stimulated to contract for 30 s at 3 Hz simulating jetting behavior and thereafter analyzed. Open bars represent unstimulated preparations; stippled bars indicate preparations that were stimulated. Text below x-axis shows additions to media. For IAA additions, preparations were incubated for 10 min prior to stimulation. N = 6 under all conditions. Box and whisker plots show the range and median value.