Developmental plasticity of cardiac anoxia-tolerance in juvenile common snapping turtles (Chelydra serpentina)

Figures

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  Figure 1.. Experimental protocol and equipment for subjecting turtle cardiomyocytes to an anoxic challenge. (a) Ventricular cardiomyocytes from juvenile turtles that embryonically developed in normoxia (21% O2) or chronic hypoxia (10% O2) were subjected to a 40-min experimental protocol to determine the effects of simulated normoxia, anoxia and reoxygenation (same treatment as normoxia) on cell shortening, [Ca2+]i, pHi and ROS production. (b) Isolated cardiomyocytes (image collected from a confocal microscope) were placed onto (c) a closed-cell bath, within (d) a custom-built chamber, in which the cardiomyocytes were subjected to the two treatments. Images courtesy of Warner Instruments (model RC-21BRFS) and Okolab (custom-built bold-line top stage incubator). (Online version in colour.)

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  Figure 2.. Effects of an anoxic challenge on juvenile turtle ventricular cardiomyocyte shortening and ion homeostasis. Cardiomyocytes were isolated from juvenile turtles that embryonically developed in either normoxia (N21) or chronic hypoxia (H10). Cardiomyocytes were exposed to simulated normoxia, anoxia and reoxygenation. (a) Cell shortening was normalized to the relaxed cell length and plotted as a percentage. (b) The absolute and (c) relative intracellular Ca2+ transient (Δ[Ca2+]i). (d) intracellular pH (pHi). (e) Reactive oxygen species (ROS) production; measured with dihydroethidium (DHE), normalized to the 5-min time-point (control) and plotted as a relative change. (f) The efficiency of cell shortening; calculated as the quotient of shortening (% relaxed cell length) divided by Δ[Ca2+]i (nM). Statistical significances are indicated by letters, for within-group differences (abN21 and ABH10; one-tailed tests used for cell shortening, pHi, and Δ[Ca2+]i), and asterisks, for between-group differences (*N21 versus H10). Values are means ± s.e.m. and significant when p ≤ 0.05. Cardiomyocyte shortening, Δ[Ca2+]i, and pHi were measured simultaneously (n = 9–10 and 10–11, for N21 and H10,…

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  Figure 3.. Effects of an anoxic challenge on juvenile turtle ventricular cardiomyocyte diastolic and systolic Ca2+ levels. Cardiomyocytes were isolated from juvenile turtles that embryonically developed in either normoxia (N21) or chronic hypoxia (H10). Representative traces of normalized Ca2+ transients from (a) N21 and (b) H10 cardiomyocytes demonstrate the effects of normoxia, anoxia and reoxygenation (see legend for colour codes) on transient shape, amplitude and duration. (c) Diastolic and (d) systolic intracellular Ca2+ ([Ca2+]i). Statistical significances are indicated by letters, for within-group differences (abN21 and ABH10), and asterisks, for between-group differences (*N21 versus H10). Values are means ± s.e.m. (n = 9–10 and 10–11, for N21 and H10, respectively) and significant when p ≤ 0.05. (Online version in colour.)

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  Figure 4.. The effect of developmental hypoxia and experimental treatment on myofilament Ca2+-sensitivity. (a,b) Correlations between the calcium transient (Δ[Ca2+]i) and cell shortening, in cardiomyocytes from juvenile turtles that embryonically developed in either normoxia (N21) or chronic hypoxia (H10). Plotted data represent normoxia (N), anoxia (A) and reoxygenation (R); N21 n-values = 26, 52 and 26 and H10 n-values = 22, 44 and 22, for normoxia, anoxia and reoxygenation, respectively). (c) Correlation between extracellular Ca2+ ([Ca2+]e) and relaxed cell length (n = 32 and 24, for N21 and H10, respectively). (Online version in colour.)