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Noncanonical EF-hand motif strategically delays Ca2+ buffering to enhance cardiac performance

Abstract

EF-hand proteins are ubiquitous in cell signaling. Parvalbumin (Parv), the archetypal EF-hand protein, is a high-affinity Ca2+ buffer in many biological systems. Given the centrality of Ca2+ signaling in health and disease, EF-hand motifs designed to have new biological activities may have widespread utility. Here, an EF-hand motif substitution that had been presumed to destroy EF-hand function, that of glutamine for glutamate at position 12 of the second cation binding loop domain of Parv (ParvE101Q), markedly inverted relative cation affinities: Mg2+ affinity increased, whereas Ca2+ affinity decreased, forming a new ultra-delayed Ca2+ buffer with favorable properties for promoting cardiac relaxation. In therapeutic testing, expression of ParvE101Q fully reversed the severe myocyte intrinsic contractile defect inherent to expression of native Parv and corrected abnormal myocardial relaxation in diastolic dysfunction disease models in vitro and in vivo. Strategic design of new EF-hand motif domains to modulate intracellular Ca2+ signaling could benefit many biological systems with abnormal Ca2+ handling, including the diseased heart.

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Figure 1: EF-hand motif design and biochemical kinetic analysis.
Figure 2: Functional screening of Parv motifs in rat adult cardiac myocytes.
Figure 3: Effects of ParvE101Q on rabbit adult cardiac myocyte contractility and relaxation.
Figure 4: Effects of ParvE101Q on Ca2+ handling and myofilaments.
Figure 5: ParvE101Q rescues depressed contraction and relaxation in failing myocytes and myocytes with induced relaxation defects.
Figure 6: Systemic rAAV delivery of ParvE101Q rescues defective relaxation in cardiomyopathy models of cell-intrinsic diastolic dysfunction in vivo.

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Acknowledgements

We thank T. Herron, E. Devaney, I. Turner, M. Maerz, F. Sjaastad, B. Liu, S. Little and T. Edwards for their assistance. We thank the Lillehei Heart Institute for support. We thank H. Sabbah (Henry Ford Hospital Heart and Vascular Institute) for providing the canine failing myocytes. We thank K.B. Andersson and G. Christensen (Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, Norway) for providing the mice with inducible cardiac myocyte–specific excision of the SERCA2a. This work was supported by the US National Institutes of Health (J.M.M. and J.D.P.).

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J.M.M. was responsible for the original concept and had a key role in study design and data interpretation. W.W. designed and conducted most experiments. M.S.B., F.I.H., M.L.A., J.D. and E.B. aided the in vivo heart function tests. E.A. generated the Ad5 and rAAV vectors for the Parv proteins. Q.L. and J.D.P. provided plasmid constructs of modified Parv. J.P.D. contributed to the measurement of Parv Ca2+ and Mg2+ dissociation rates. J.M.M. and W.W. prepared the manuscript with contributions from J.D.P. and J.P.D.

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Correspondence to Joseph M Metzger.

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Wang, W., Barnabei, M., Asp, M. et al. Noncanonical EF-hand motif strategically delays Ca2+ buffering to enhance cardiac performance. Nat Med 19, 305–312 (2013). https://doi.org/10.1038/nm.3079

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