Abstract 493: Designer Ca2+ Buffers for Enhancing Contractile and Relaxation Performance in Adult Rabbit Cardiac Myocytes
Background: The cytosolic Ca2+ binding protein parvalbumin (Parv) is a unique delayed Ca2+ buffer that improves cardiac diastolic function under normal and disease conditions. The potential of wild-type Parv as a possible therapeutic strategy to redress diastolic heart failure is limited by its inhibition of contractility due to partial Ca2+ buffering in systole.
Hypothesis: Targeted genetic engineering of the Ca2+ and Mg2+ binding sites of Parv will alter Ca2+ buffering characteristics to limit contractility deficits.
Methods/Results: Several point mutations of wild-type β-Parv that yield variations in Ca2+/Mg2+ binding affinities were generated. After adenovirus-mediated acute gene transfer, these mutants showed time-dependent expression and effects on myocytes relaxation. The Parv D51A/E101Q/F102W (Parv-DEF) mutant, which has 100 fold lowered Ca2+ binding affinity and doubled Mg2+ binding affinity, significantly accelerated the relaxation of adult rabbit cardiac myocytes. Interestingly, in distinction from wild type Parv, Parv-DEF enhanced the contractility of rabbit myocytes (sarcomere length shortening: 33.4±3.6 nm vs. 57.8±6.2 nm in control and Parv-DEF transferred cells, respectively, n=40–58, P<0.001). Further, the Parv-DEF mutant accelerated the Ca2+ transient decay (time from peak to 50% decay: 627±26 ms vs. 474±27 ms in control and Parv-DEF transferred cells, respectively, n=43–46, P<0.001) without influencing its amplitude (Ca2+ transient amplitude (360/380 nm ratio of fura-2): 0.059±0.004 vs. 0.056±0.003 in control and Parv-DEF transferred cells, respectively, n=43–46). Parv-DEF gene transfer did not influence sarcoplasmic reticulum Ca2+ content or the responsiveness to β adrenergic stimulation of rabbit cells. With increased pacing frequency, Parv-DEF-induced positive lusitropic effect was reduced but the positive inotropic effect persisted.
Conclusion: Parv-DEF specifically buffers Ca2+ at the diastolic phase to speed up relaxation and may also exert Ca2+-independent effects to enhance contractility in rabbit cardiac myocytes. The positive lusitropic and contractile effects of Parv-DEF make this designer Ca2+ buffer a promising candidate for the treatment of diastolic heart failure.