High calcium and dobutamine positive inotropy in the perfused mouse heart: myofilament calcium responsiveness, energetic economy, and effects of protein kinase C inhibition

Abstract

Background: In perfused hearts, high calcium-induced inotropy results in less developed pressure relative to myocardial oxygen consumption compared to the beta-adrenergic agonist dobutamine. Calcium handling is an important determinant of myocardial oxygen consumption. Therefore, we hypothesized that this phenomenon was due to reduced myofilament responsiveness to calcium, related to protein kinase C activation.

Results: Developed pressure was significantly higher with dobutamine compared to high perfusate calcium of 3.5 mM (73 +/- 10 vs 63 +/- 10 mmHg, p < 0.05), though peak systolic intracellular calcium was not significantly different, suggesting reduced myofilament responsiveness to intracellular calcium with high perfusate calcium. The ratio of developed pressure to myocardial oxygen consumption, an index of economy of contraction, was significantly increased with dobutamine compared to high perfusate calcium (1.35 +/- 0.15 vs 1.15 +/- 0.15 mmHg/micromoles/min/g dry wt, p < 0.05), suggesting energetic inefficiency with high perfusate calcium. The specific protein kinase C inhibitor, chelerythrine, significantly attenuated the expected increase in developed pressure when increasing perfusate calcium from 2.5 to 3.5 mM (3.5 mM: 64 +/- 8 vs 3.5 mM + chelerythrine: 55 +/- 5 mmHg, p < 0.05), though had no effects on dobutamine, or lower levels of perfusate calcium (1.5 to 2.5 mM).

Conclusions: By measuring intracellular calcium, developed pressures and myocardial oxygen consumption in perfused mouse hearts, these results demonstrate that high perfusate calcium positive inotropy compared to dobutamine results in reduced myofilament responsiveness to intracellular calcium, which is associated with energetic inefficiency and evidence of protein kinase C activation.

Figure 1

A. Developed pressure at perfusate calcium 1.5 – 3.5 mM and with dobutamine (dob, 0.9 μM). B. Peak systolic intracellular calcium, C. Myocardial oxygen consumption (MVO₂), D. Ratio of developed pressure to myocardial oxygen consumption (DP/ MVO₂). Symbols for Figure 1A: ^* p < 0.005 vs perfusate calcium 2.0, 2.5, 3.5 mM and dobutamine; † p < 0.05 vs dobutamine and perfusate calcium 3.5 mM; §p < 0.0001 vs dobutamine; ^** p < 0.05 vs dobutamine; Symbols for Figure 1B:^* p < 0.005 vs perfusate calcium 3.5 mM and dobutamine; † p < 0.05 vs perfusate calcium 3.5 mM and dobutamine; Symbols for Figure 1C:^† p = 0.05 vs dobutamine; ^§ p < 0.05 vs dobutamineSymbols for Figure 1D:^* p < 0.0001 vs perfusate calcium 2.0, 2.5, 3.5 mM and dobutamine; † p < 0.0001 vs dobutamine; §p < 0.0005 vs dobutamine; ^** p < 0.05 vs dobutamine

Figure 2

Example of normalized pressure waveforms for dobutamine and 3.5 mM perfusate calcium, illustrating abbreviation of the dobutamine waveform compared to 3.5 mM perfusate calcium.

Figure 3

A: Averaged calcium transients (6–8 cycles) at perfusate calcium 1.5 and 2.5 mM, B: perfusate calcium 2.5 and 3.5. mM, and C: perfusate calcium 2.0 mM and with dobutamine. D. Phase-plane plots of pressure and calcium at perfusate calcium 1.5 and 2.5 mM, E: perfusate calcium 2.5 and 3.5. mM, and F: perfusate calcium 2.0 mM and with dobutamine.

Figure 4

Averaged phase-plane plots for high perfusate calcium and dobutamine. While the increase in intracellular calcium is similar for both, at peak pressure the ratio of pressure to intracellular calcium is significantly higher with dobutamine (^* p < 0.05).

Figure 5

A. Effects of protein kinase C inhibitor (PKCi) chelerythrine on inotropic responses (developed pressure) of perfusate calcium 1.5 to 2.5 mM (left), 2.5 to 3.5 mM (middle), and dobutamine (right). B. Effects of PKCi with perfusate calcium 2.5 to 3.5 mM (left) on peak systolic intracellular calcium, and basal diastolic calcium (right). Baseline controls values (perfusate calcium 1.5, 2.0 or 2.5 mM) for these experiments include those experiments with addition of chelerythrine to the higher perfusate calcium or dobutamine after control measurements, combined also with those experiments without chelerythrine. ^* p < 0.05, and ^** p < 0.005.