High-Throughput Tissue Bioenergetics Analysis Reveals Identical Metabolic Allometric Scaling for Teleost Hearts and Whole Organisms

Abstract

Organismal metabolic rate, a fundamental metric in biology, demonstrates an allometric scaling relationship with body size. Fractal-like vascular distribution networks of biological systems are proposed to underlie metabolic rate allometric scaling laws from individual organisms to cells, mitochondria, and enzymes. Tissue-specific metabolic scaling is notably absent from this paradigm. In the current study, metabolic scaling relationships of hearts and brains with body size were examined by improving on a high-throughput whole-organ oxygen consumption rate (OCR) analysis method in five biomedically and environmentally relevant teleost model species. Tissue-specific metabolic scaling was compared with organismal routine metabolism (RMO2), which was measured using whole organismal respirometry. Basal heart OCR and organismal RMO2 scaled identically with body mass in a species-specific fashion across all five species tested. However, organismal maximum metabolic rates (MMO2) and pharmacologically-induced maximum cardiac metabolic rates in zebrafish Danio rerio did not show a similar relationship with body mass. Brain metabolic rates did not scale with body size. The identical allometric scaling of heart and organismal metabolic rates with body size suggests that hearts, the power generator of an organism’s vascular distribution network, might be crucial in determining teleost metabolic rate scaling under routine conditions. Furthermore, these findings indicate the possibility of measuring heart OCR utilizing the high-throughput approach presented here as a proxy for organismal metabolic rate—a useful metric in characterizing organismal fitness. In addition to heart and brain OCR, the current approach was also used to measure whole liver OCR, partition cardiac mitochondrial bioenergetic parameters using pharmacological agents, and estimate heart and brain glycolytic rates. This high-throughput whole-organ bioenergetic analysis method has important applications in toxicology, evolutionary physiology, and biomedical sciences, particularly in the context of investigating pathogenesis of mitochondrial diseases.

Cost of Tolerance: Physiological Consequences of Evolved Resistance to Inhabit a Polluted Environment in Teleost Fish Fundulus heteroclitus

“…29 Accordingly reduced AS in ER fish compared to KC at 34 °C may affect their capacity to survive thermally fluctuating habitats. Furthermore, cardiac energy metabolism is thought to be causally linked to cardiac output and organismal metabolism, 28,42 and cardiac mitochondrial dysfunction at warmer temperatures is associated with teleost thermal limits. 24 Accordingly, reduced cardiac mitochondrial spare capacity and organismal aerobic capacity observed at 34 °C in AW fish may significantly contribute to the decrease in upper thermal tolerance of AW and other ER fish.…”

“…XF e Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA) was used for heart OCR measurements in KC and AW fish (collected in 2014) as previously described 28 at 24 °C ( n = 33) and 34 °C ( n = 19–21). Basal OCR measurements were followed by an injection of 5 μ M carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) (Sigma-Aldrich, St. Louis, MO) to obtain maximal respiration.…”

“…Data are presented as OCR per 1 mg of heart by correcting for heart size according to Y ∞ M b , where Y is metabolic rate, M is heart mass, and b is the heart specific scaling exponent for basal cardiac OCR; b = 0.6588 for F. heteroclitus. 28 The same exponent was applied for normalizing FCCP-induced maximal rates, although it is possible this value is lower considering previous data on zebrafish Danio rerio. 28 If the exponent is lower it would lead to even higher estimation of maximal rates for AW fish, thus the current approach is likely to be more conservative.…”

“…28 The same exponent was applied for normalizing FCCP-induced maximal rates, although it is possible this value is lower considering previous data on zebrafish Danio rerio. 28 If the exponent is lower it would lead to even higher estimation of maximal rates for AW fish, thus the current approach is likely to be more conservative. Spare capacity (i.e., mitochondrial scope) is the difference between maximal and basal OCR.…”

A bioenergetics assay for studying the effects of environmental stressors on mitochondrial function in vivo in zebrafish larvae

“…However, recent technological advances, such as the development of the XF e 24 Extracellular Flux Analyzer (Agilent Technologies, Santa Clara, CA, USA), have allowed for a more streamlined method to analyze mitochondrial respiration using a 24- or 96-well microplate format. Despite significant advancements in development and application of assays that evaluate mitochondrial function in vitro and ex vivo (Jayasundara et al, 2015a; Tiernan et al, 2015; Wills et al, 2015), there is an ongoing need to develop medium- to high-throughput assays that rapidly assess mitochondrial function within whole organisms to ensure physiologically-relevant responses are measured. Therefore, the goals of this study were to 1) develop and optimize a reliable, robust assay using the XF e 24 Extracellular Flux Analyzer and pharmacological agents to obtain in vivo measurements of mitochondrial respiratory chain parameters in zebrafish larvae, and 2) apply this assay to examine how developmental exposure to subteratogenic concentrations of three PAHs – benzo(a)pyrene, phenanthrene, and fluoranthene - affects normal mitochondrial function.…”