Abstract
Parvalbumin (PV), an EF-hand protein family member, is a delayed calcium buffer that exchanges magnesium for calcium to facilitate fast skeletal muscle relaxation. Genetic approaches that express parvalbumin in the heart also enhance relaxation and show promise of being therapeutic against various cardiac diseases where relaxation is compromised. Unfortunately, skeletal muscle PVs have very slow rates of Ca2+ dissociation and are prone to becoming saturated with Ca2+, eventually losing their buffering capability within the constantly beating heart. In order for PV to have a more therapeutic potential in the heart, a PV with faster rates of calcium dissociation and high Mg2+ affinity is needed. We demonstrate that at 35°C, rat β-PV has an ~30-fold faster rate of Ca2+ dissociation compared to rat skeletal muscle α-PV, and still possesses a physiologically relevant Ca2+ affinity (~100 nM). However, rat β-PV will not be a delayed Ca2+ buffer since its Mg2+ affinity is too low (~1 mM). We have engineered two mutations into rat β-PV, S55D and E62D, when observed alone increase Mg2+ affinity up to fivefold, but when combined increase Mg2+ affinity ~13-fold, well within a physiologically relevant affinity. Furthermore, the Mg2+ dissociation rate (172/s) from the engineered S55D, E62D PV is slow enough for delayed Ca2+ buffering. Additionally, the engineered PV retains a high Ca2+ affinity (132 nM) and fast rate of Ca2+ dissociation (64/s). These PV design strategies hold promise for the development of new therapies to remediate relaxation abnormalities in different heart diseases and heart failure.
Highlights
Diastolic dysfunction, the inability of the heart to properly relax, is a hallmark of many heart diseases and heart failure (Periasamy and Janssen, 2008)
One novel approach to counter the Ca2+ imbalance has been borrowed from a specialized mechanism in fast twitch skeletal muscle that aids in relaxation
In order to simplify the purification protocol for PV, we speculated that unlike other proteins, it would not denature and precipitate in 100% saturating Ammonium sulfate (AMS) when in the presence of Ca2+ and Mg2+. Consistent with this idea, 100% AMS saturation precipitated most the bacterial proteins, leaving PV and nucleic acids in the supernatant as judged by Coomassie Brilliant Blue and Ethidium Bromide staining as can be seen in Figures 1A,B
Summary
The inability of the heart to properly relax, is a hallmark of many heart diseases and heart failure (Periasamy and Janssen, 2008) During this condition, it is generally thought that the cardiac myocyte loses the ability to efficiently and effectively manage intracellular Ca2+, prolonging relaxation (Bers, 2006; van der Velden, 2011). One novel approach to counter the Ca2+ imbalance has been borrowed from a specialized mechanism in fast twitch skeletal muscle that aids in relaxation. This mechanism utilizes a protein called parvalbumin (PV) to achieve faster relaxation in combination with the sarcoplasmic reticulum Ca2+ ATPase (Hou et al, 1993). In a relaxed muscle, PV is bound with Mg2+ and cannot bind
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