Cardiac gene delivery of parvalbumin (Parv), an EF-hand Ca2+ buffer, has been studied as a therapeutic strategy for diastolic heart failure, in which slow Ca2+ reuptake is an important contributor. A limitation of wild-type (WT) Parv is the significant trade-off between faster relaxation and blunted contraction amplitude, occurring because WT-Parv sequesters Ca2+ too early in the cardiac cycle and prematurely truncates sarcomere shortening in the facilitation of rapid relaxation. We recently demonstrated that an E → Q substitution (ParvE101Q) at amino acid 12 of the EF-hand Ca2+/Mg2+ binding loop disrupts bidentate Ca2+ binding, reducing Ca2+ affinity by 99-fold and increasing Mg2+ affinity twofold. ParvE101Q caused faster relaxation and not only preserved contractility, but unexpectedly increased it above untreated myocytes. To gain mechanistic insight into the increased contractility, we focused here on amino acid 12 of the EF-hand motif. We introduced an E → D substitution (ParvE101D) at this site, which converts bidentate Ca2+ coordination to monodentate coordination. ParvE101D decreased Ca2+ affinity by 114-fold and increased Mg2+ affinity 28-fold compared to WT-Parv. ParvE101D increased contraction amplitude compared to both untreated myocytes and myocytes with ParvE101Q, with limited improvement in relaxation. Additionally, ParvE101D increased spontaneous contractions after pacing stress. ParvE101D also increased Ca2+ transient peak height and was diffusely localized around the Z-line of the sarcomere, suggesting a Ca2+-dependent mechanism of enhanced contractility. Sarcoplasmic reticulum Ca2+ load was not changed with ParvE101D, but postpacing Ca2+ waves were increased. Together, these data show that inverted Ca2+/Mg2+ binding affinities of ParvE101D increase myocyte contractility through a Ca2+-dependent mechanism without altering sarcoplasmic reticulum Ca2+ load and by increasing unstimulated contractions and Ca2+ waves. ParvE101D provides mechanistic insight into how changes in the Ca2+/Mg2+ binding affinities of parvalbumin's EF-hand motif alter function of cardiac myocytes. These data are informative in developing new Ca2+ buffering strategies for the failing heart.
Bibliographical noteFunding Information:
This research was supported by funds from National Institutes of Health (NIH) to J.M.M. and by NIH National Heart, Lung, and Blood Institute (NHLBI) award 5F32HL115876 to M.L.A.