Mechanisms of enhanced cardiorespiratory performance under hyperoxia differ with exposure duration in yellowtail kingfish

Hyperoxia has been shown to expand the aerobic capacity of some fishes, although there have been very few studies examining the underlying mechanisms and how they vary across different exposure durations. Here, we investigated cardiorespiratory function of yellowtail kingfish (Seriola lalandi) acutely (~20 hours) and chronically (3-5 weeks) acclimated to hyperoxia (~200 % air saturation). Our results show that aerobic performance of kingfish is limited in normoxia and increases with environmental hyperoxia. Aerobic scope was elevated in both hyperoxia treatments driven by a ~33% increase in maximum O2 uptake (MO2max), although the mechanisms differed across treatments. Fish acutely transferred to hyperoxia primarily elevated tissue O2 extraction, while increased stroke volume-mediated maximum cardiac output was the main driving factor in chronically acclimated fish. Still an improved O2 delivery to the heart in chronic hyperoxia was not the only explanatory factor as such. Here, maximum cardiac output only increased in chronic hyperoxia compared to normoxia when plastic ventricular growth occurred, as increased stroke volume was partly enabled by an ~8-12% larger relative ventricular mass. Our findings suggest that hyperoxia may be used long-term to boost cardiorespiratory function potentially rendering fish more resilient to metabolically challenging events and stages in their life-cycle.

Methods
We recorded the rate of whole-animal O2 uptake (MO2) using intermittent-flow respirometry where the % air saturation inside the respirometer was continuously measured using an O2 optode connected to a Firesting O2 system. Automated flush pumps, were set to flush the respirometers for 5 min every 7 min (i.e., 2 min measurement cycles). MO2 was calculated from the slope of the decline in % air saturation between flushes using the following formula: MO2 = (Vr – Vf) × (Δ%Sat/t) × α; where Vr is the volume of the respirometer, Vf is the volume of the fish assuming that 1 g of tissue equals 1 ml of water, Δ%Sat/t is the change in % O2 saturation per time and α is the temperature-, salinity- and atmospheric pressure-dependent solubility coefficient of O2. The first ~30 s of each measurement cycle was excluded from the slope determination to ensure the inclusion of only the linear section of the decline in O2. SMR (standard metabolic rate) was calculated as the mean of the lowest 20% of all MO2 values obtained throughout the whole 20+ hours of recordings, with measurements 2 standard deviations below the mean of the lowest 20% removed as outliers. MO2max (maximum O2 uptake) was calculated as the highest MO2 value obtained at any point following exercise. The lowest 20% were chosen instead of the more commonly used lowest 10% to maximize the number of fish that achieve EPOC repayment for each treatment. Aerobic scope was then calculated as the difference between SMR and MO2max⁠. EPOC (excess post-exercise O2 consumption) was calculated as the area between the MO2 curve following the stress protocol and SMR + 10% using GraphPad Prism 9.1.2. Briefly, before analysis, individual MO2 traces were smoothed by removing routine MO2 values that were 10% larger than the previous value. EPOC duration was defined as the time in hours between the exhaustive protocol and the intersection of the MO2 trace with the individual SMR + 10%. The rate of EPOC repayment was defined as EPOC/EPOC duration. We cleaned the respirometers thoroughly after each trial, and measured background respiration before and after each individual experiment and was negligible throughout the study (< 0.2% of the MO2 slope).

Heart rate was calculated from the pulsating blood flow signal, and stroke volume was calculated as cardiac output/heart rate. The arterial-venous O2 difference was estimated as MO2/cardiac output (Fick´s principle´s equation). All cardiovascular variables were measured simultaneously with MO2 recordings, and cardiovascular variables measured concomitantly to SMR, MO2max and aerobic scope are referred to henceforth as resting, maximum and scope. Additionally, cardiorespiratory dynamics were assessed immediately prior to and at six time points following the exhaustive protocol and thus comprised: pre-exhaustion values (average of two last cycles prior to exhaustive protocol), immediately after the exhaustive protocol (0 h), and 0.5, 1, 2, 3 and 5 h following the exhaustive protocol. All measurements were derived from the average of two MO2 cycles, except for the 0 h value, which was taken during the first measurement immediately after the exhaustive protocol. Additionally, peak cardiorespiratory responses (i.e., the highest arterial-venous O2 content difference, cardiac output, stroke volume and heart rate measured at any time point throughout the recovery period independently from MO2max) and time to peak cardiorespiratory responses (i.e., the time elapsed from the beginning of the cardiorespiratory measurements following exhaustive exercise to the peak responses) were determined for each fish.

The relative ventricular mass was calculated as wet mass of the ventricle/body mass × 100. To determine the relative % of ventricular compact myocardium, the spongy and compact layers were separated, dried and weighed⁠. The percentage compact myocardium was calculated as dry mass of compact myocardium/dry mass of ventricle × 100. The relative spleen mass was calculated as wet mass of the spleen/body mass × 100.

已有研究表明，高氧（hyperoxia）可提升部分鱼类的有氧能力，但针对其潜在调控机制及不同暴露时长下的差异的相关研究仍较为匮乏。本研究以黄尾鰤（Seriola lalandi）为研究对象，分别对其开展急性（约20小时）与慢性（3~5周）高氧（约200%空气饱和度）驯化实验，探究其心肺功能变化。结果显示，黄尾鰤的有氧代谢性能在常氧（normoxia）条件下存在上限，且随环境高氧水平升高而显著增强。两种高氧处理组的有氧代谢范围均有所提升，这源于最大摄氧量（maximum O2 uptake, MO2max）升高约33%，但两组的调控机制存在显著差异：急性转入高氧环境的鱼类主要通过提升组织摄氧能力实现有氧性能优化，而慢性驯化组的核心调控因子为每搏输出量（stroke volume）介导的最大心输出量（cardiac output）升高。值得注意的是，慢性高氧条件下心脏氧输送能力的提升并非唯一解释因素：仅当发生心室可塑性生长时，慢性高氧组的最大心输出量才较常氧组有所提升，这是因为每搏输出量的提升部分得益于相对心室质量（relative ventricular mass）增加8%~12%。本研究结果提示，高氧可长期用于提升鱼类心肺功能，从而增强其应对代谢应激事件及生命周期关键阶段的抗逆能力。

## 材料与方法
本研究采用间歇流呼吸测量法（intermittent-flow respirometry）记录整体动物摄氧率（MO2），呼吸室内的空气饱和度通过连接至Firesting氧气分析系统（Firesting O2 system）的光学溶解氧传感器（O2 optode）持续监测。自动冲洗泵设置为每7分钟冲洗呼吸室5分钟（即每测量周期为2分钟）。MO2通过冲洗间隔内空气饱和度的下降斜率计算得到，计算公式为：MO2 = (Vr – Vf) × (Δ%Sat/t) × α；其中Vr为呼吸室体积，Vf为鱼体体积（假设1g组织对应1ml水），Δ%Sat/t为单位时间内氧气饱和度的变化量，α为受温度、盐度和大气压影响的氧气溶解系数。每轮测量周期的前约30秒被排除在斜率计算之外，以确保仅纳入氧浓度下降的线性阶段。标准代谢率（standard metabolic rate, SMR）通过全时段20小时以上的记录中最低20%的MO2值的平均值计算得到，同时将低于最低20%平均值2个标准差的数值作为异常值剔除。最大摄氧量（MO2max）为运动后任一时刻测得的最高MO2值。本研究选择最低20%的数值而非更常用的最低10%，以最大化各处理组中完成运动后过量氧耗（excess post-exercise O2 consumption, EPOC）恢复的个体数量。有氧代谢范围通过SMR与MO2max的差值计算得到。采用GraphPad Prism 9.1.2软件计算EPOC，即运动后MO2曲线与SMR+10%区间围成的面积。具体步骤如下：分析前，先对个体MO2轨迹进行平滑处理，剔除较前一数值大10%的常规MO2值。EPOC持续时间定义为力竭运动方案实施后至MO2轨迹与个体SMR+10%曲线相交的时长（小时）；EPOC恢复速率定义为EPOC总量与EPOC持续时间的比值。每次试验结束后彻底清洁呼吸室，并在每个个体实验前后测量本底呼吸速率，本研究中本底呼吸可忽略不计（低于MO2斜率的0.2%）。

心率通过搏动血流信号计算得到，每搏输出量通过心输出量除以心率计算得到。动静脉氧差通过MO2除以心输出量估算（费克原理公式）。所有心血管变量与MO2记录同步测量，与SMR、MO2max及有氧代谢范围对应的心血管变量分别记为静息、最大及代谢范围相关变量。此外，本研究在力竭运动方案实施前即刻及实施后6个时间点评估心肺动力学变化，包括：力竭前数值（力竭前最后2个测量周期的平均值）、力竭后即刻（0小时），以及力竭后0.5、1、2、3和5小时。除0小时数值取自力竭后首个测量周期外，其余测量值均取自2个MO2测量周期的平均值。同时，本研究还测定了每条鱼的心肺峰值响应（即恢复期内任一时刻测得的最高动静脉氧差、心输出量、每搏输出量及心率，与MO2max无关）及心肺峰值响应达峰时间（即从力竭运动后开始心肺测量至达到峰值响应的时长）。

相对心室质量通过心室湿重/体质量×100计算得到。为测定心室致密心肌层的相对占比，将心肌的海绵层与致密层分离、干燥并称重，致密心肌层占比通过致密层干重/心室干重×100计算得到。相对脾脏质量通过脾脏湿重/体质量×100计算得到。