Unilateral Acute Renal Ischemia-Reperfusion Injury Induces Cardiac Dysfunction through Intracellular Calcium Mishandling

Figures

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  Figure 1. Contractility of electrically-stimulated cardiomyocytes after renal ischemia and reperfusion. (A) Cell contraction profiles of cardiomyocytes from sham-treated mice (black line) and from mice subjected to ischemia and reperfusion (I/R) for 7 (red line) and 15 (red discontinuous line) days. (B–D) Cell contraction (B), shortening (C) and re-lengthening (D) velocities. Cardiomyocytes were electrically stimulated at 2 Hz. Data are expressed as mean ± SEM. ** p < 0.01, *** p < 0.001 versus sham, ### p < 0.001 versus 7 d I/R; sham (n = 73 cells, N = 10 mice), 7 d I/R (n = 39 cells, N = 10 mice) and 15 d I/R (n = 90 cells, N = 6 mice).

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  Figure 2. Systolic Ca2+ transient release in electrically stimulated cardiomyocytes. (A) Representative line-scan images of cardiomyocytes from sham-treated mice and from mice subjected to ischemia and reperfusion (I/R) for 7 and 15 days. Cardiomyocytes were electrically stimulated at 2 Hz. (B) Mean peak fluorescence Ca2+ transients (F/F0). (C) Decay time constant, Tau (ms). Data are expressed as mean ± SEM. *** p < 0.001 versus sham, ### p < 0.001 versus 7 d I/R; sham (n = 80 cells, N = 10 mice), 7 d I/R (n = 61 cells, N = 10 mice), and 15 d I/R (n = 91 cells, N = 6 mice). (D) Representative immunoblots of SERCA2a and GAPDH and (E) quantification of all experiments corresponding to cardiac protein expression. Data are presented as fold of change of sham levels ± SEM (sham = 9; 7 d I/R = 8; 15 d I/R = 8). * p < 0.05 versus sham.

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  Figure 3. Sarcoplasmic reticulum Ca2+ load analysis on electrically stimulated cardiomyocytes. (A) Representative line-scan images of caffeine-evoked Ca2+ transients in cardiomyocytes from sham-treated mice and from mice subjected to ischemia and reperfusion (I/R) for 7 and 15 days. Cardiomyocytes were electrically stimulated at 2 Hz. (B) Mean values of the transient amplitude evoked by caffeine (peak fluorescence calcium, F/F0) in stimulated cardiomyocytes. (C) Decay time constant, Tau, in milliseconds. Data are expressed as mean ± SEM. * p < 0.05 versus sham, ## p < 0.01 versus 7 d I/R; sham (n = 37 cells, N = 10 mice), 7 d I/R (n = 59 cells, N = 10 mice), and 15 d I/R (n = 73 cells, N = 6 mice).

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  Figure 4. Diastolic Ca2+ leak in cardiomyocytes after renal I/R. (A) Representative line-scan images of Ca2+ sparks in quiescent cardiomyocytes from sham-treated mice and from mice subjected to ischemia and reperfusion (IR) for 7 and 15 days. (B) Frequency of Ca2+ sparks (presented in n·s−1·100 µm−1). (C) Mean peak of fluorescence Ca2+ sparks (F/F0). (D) Full width at half maximum of fluorescence of sparks (µm). (E) Full duration at half maximum of fluorescence Ca2+ sparks (ms). Data are expressed as mean ± SEM. * p < 0.05, *** p < 0.001 versus sham; # p < 0.05, ## p < 0.01 versus and ### p < 0.001 versus 7d I/R. sham (n = 57 cells, N = 10 mice), 7 d I/R (n = 43 cells, N = 10 mice) and 15 d I/R (n = 85 cells, N = 6 mice).

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  Figure 5. Spontaneous Ca2+ release in cardiomyocytes after renal I/R. (A) Representative line-scan images of cardiomyocytes from mice subjected to ischemia and reperfusion (I/R) for 7 and 15 days. (B) Spontaneous Ca2+ release measured as pro-arrhythmogenic events in cardiomyocytes. Data are presented as % of total analyzed cells. Data are expressed as mean of continuous variables after Fisher test. ** p < 0.01, *** p < 0.001 versus sham; sham (n = 56 cells, N = 4 mice), 7d I/R (n = 50 cells, N = 3 mice), 15d I/R (n = 99 cells, N = 6 mice). In this protocol each cell was recorded 8 consecutive times for 1.5 s. Statistical analyses were performed per scanned-file.

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  Figure 6. Pro-arrhythmogenic events in electrically stimulated cardiomyocytes. (A) Representative line-scan images and contraction profiles of cardiomyocytes from sham-treated mice and from mice subjected to ischemia and reperfusion (IR) for 7 and 15 days. Cardiomyocytes were electrically stimulated at 2 Hz in 7-pulse sequences. * Represents arrhythmic Ca2+ events (spontaneous abnormal release of Ca2+ as waves or automatic contractions). (B) Proarrhythmogenic Ca2+ release following the arrhythmia protocol in cardiomyocytes. Data presented as % of total analyzed cells with events. Data are expressed as mean of continuous variables after Fisher test. *** p < 0.001 versus sham; sham (n = 113 cells, N = 5 mice), 7d I/R (n = 77 cells, N = 7 mice), 15d I/R (n = 150 cells, N = 6 mice). In this protocol, each cell was recorded once for 30 s. Statistical analyses were performed per cell.

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  Figure 7. Schematic drawing of the take-home messages.