Figure 1.
Experimental timeline and SERCA2 protein determination in FL and KO hearts.
A, Experimental Timeline. All animals were injected with tamoxifen then sacrificed either 1 or 4 weeks later for heart isolation. KO mice expressed the αMHC-MerCreMer transgene, which efficiently excised LoxP-flanked exons of the Serca2 locus in response to tamoxifen. FL mice received tamoxifen, but retained normal SERCA2 levels due to lack of MerCreMer. B-C, at 1 week post-tamoxifen, SERCA2 content in KO hearts was diminished to 32% of FL. D-E, at 4 weeks post-tamoxifen, faint SERCA2 bands were observed in 20 µg of heart lysate. ***: P<0.001 for FL vs. KO at either 1 week or 4 weeks post-tamoxifen injection, as determined by unpaired two-tailed t test.
Figure 2.
Langendorff protocol and individual LV pressure traces.
A, Langendorff Protocol. After initial equilibration, pacing frequency was stepped between 3 and 12 Hz in 1-Hz intervals. After pacing at 12 Hz, hearts were re-equilibrated for 10 minutes in KHB lacking pyruvate and then subjected to 20 minutes no-flow ischemia and 60 minutes reperfusion. B, Representative traces of LV pressure from 1-week FL (solid line), 1-week KO (dashed line), and 4-week KO (dotted line) hearts sampled over 0.6 seconds. C, Individual peaks normalized to developed pressure. Note the differences in time scale for each normalized peak. i, ii, and iii: 3, 7, and 12 Hz traces, respectively, for either absolute LV pressure (B) or LV pressure normalized to own developed pressure (C).
Table 1.
Animal Characteristics.
Table 2.
Baseline Isolated Heart Function.
Figure 3.
Summary of LV Developed Pressure (LVDP) in isolated Serca2fl/fl and KO hearts.
A, 1-week KO vs. 1-week FL LVDP across a wide stimulation frequency range (3–12 Hz). B, 4-week KO vs. 4-week FL LVDP. Both 1-week and 4-week FL hearts demonstrated a negative staircase force-frequency response as pacing frequency increased from 7 Hz baseline. 1-week KO vs. 4-week KO: P<0.05 at all pacing frequencies. Some FL hearts could not be paced at very low frequencies: for 1-week FL, the 3 and 4 Hz data points represent N = 3 observations, not N = 4 for all other pacing frequencies. For 4-week FL, 3 Hz represents N = 3 observations, and 4 Hz represents N = 4 observations, rather than N = 5 for all other pacing frequencies. *: P<0.05; **: P<0.01; ***: P<0.001 for FL vs. KO at either 1 week or 4 weeks post-tamoxifen injection, as determined by two-way ANOVA with Bonferroni post-test. Symbols are mean ± SEM.
Figure 4.
Summary of LV End-Diastolic Pressures (LVEDP) in isolated Serca2fl/fl and KO hearts.
A, LVEDP of 1-week FL and KO hearts from 3–12 Hz stimulation frequency. B, LVEDP of 4-week FL and KO hearts from 3–12 Hz stimulation frequency. KO hearts at both 1 and 4 weeks post-tamoxifen injection underwent a pronounced increase in LVEDP as stimulation frequency increased. *: P<0.05; **: P<0.01; ***: P<0.001 for FL vs. KO at either 1 week or 4 weeks post-tamoxifen injection, as determined by two-way ANOVA with Bonferroni post-test.
Figure 5.
Time to 50% Relaxation (T50R), Full-Duration at Half-Maximum (FDHM), Peak Width, and Rest Time in isolated Serca2fl/fl and KO hearts.
A, T50R; B, FDHM; C, Peak Width; and D, Rest Time in 1-week FL, 1-week KO, 4-week FL, and 4-week KO hearts across 3–12 Hz stimulation frequencies. T50R (A) is the time (in ms) required for pressure to decay from peak to 50% of peak. FDHM (B) is the sum of the time to 50% pressure rise and the time to 50% pressure relaxation and indicates time spent in the contracted state. Peak Width (C) is the time duration between initiation of contraction and return to baseline. C, dashed line indicates the calculated pacing cycle length in milliseconds (PCL = 1000 ms s−1 / Pacing Frequency s−1) to compare the contractile cycle length to the maximum possible peak width at each pacing frequency. Rest Time (D) is the difference between calculated PCL and Peak Width. Symbols indicate mean ± SEM. #: P<0.05 FL vs KO (1-week). §: P<0.05 FL vs KO (4-week). †: P<0.05 1-week KO vs 4-week KO.
Figure 6.
LV Developed Pressures (LVDP) and LVDP normalized to baseline performance during ischemia-reperfusion injury in Serca2fl/fl and KO hearts.
A, LVDP of 1-week FL and 1-week KO hearts at baseline, during ischemia (black bar, minutes 1–20), and during reperfusion (gray bar, minutes 30–80). B, as in (A) except all values normalized to each heart's baseline LVDP. C and D, as with (A) and (B) for 4-week FL and 4-week KO hearts. Baseline values collected immediately prior to the onset of ischemia. One 1-week FL heart encountered a bubble in the perfusion line between pacing and ischemia-reperfusion and became infarcted, so 1-week FL N = 3 for ischemia-reperfusion (Figures 6 and 7). Symbols indicate mean ± SEM. No significant differences between FL and KO at 1 week or 4 weeks post-tamoxifen injection were found by two-way ANOVA.
Figure 7.
LV End-Diastolic Pressures (LVEDP) during ischemia-reperfusion injury in Serca2fl/fl and KO hearts.
A, LVEDP of 1-week FL and KO hearts at baseline, during ischemia (black bars, minutes 1–20), and during reperfusion (gray bar, minutes 30–80). B, as (A) for 4-week FL and KO hearts. FL and KO groups were not significantly different (P>0.05) at 1 or 4 weeks post-KO, by two-way ANOVA.
Figure 8.
Caffeine perfusion of isolated 4-week FL and KO hearts.
A, Representative traces of LV pressure from FL and KO hearts spanning the final minute of baseline recording and the first nine minutes of caffeine perfusion. B, LVDP of 4-week FL (N = 6) and KO (N = 12) hearts upon perfusion with 10 mM caffeine. Washout with normal KHB began after 10 minutes of caffeine perfusion and proceeded for 20 minutes. Baseline (“BL”) indicates heart performance at the fifth minute of baseline recording, just prior to caffeine perfusion. Dead space in perfusion line took about 1 minute to clear at both switch timepoints. C, LVEDP of 4-week FL and KO hearts upon perfusion with 10 mM caffeine. D, LV End-Systolic Pressure (LVESP) of 4-week FL and KO hearts upon perfusion with 10 mM caffeine, normalized to baseline level. E, Time of survival for 4-week FL and KO hearts from beginning of caffeine perfusion until first 15-second window in which contractile frequency was outside 7±0.5 Hz. Log-rank test: P<0.05 between FL and KO groups. Icons indicate mean, and error bars indicate SEM unless smaller than icon. Panels B, C, and D analyzed by two-way ANOVA. Brackets indicate results of Bonferroni post-tests.