Over five million people in the U.S. suffer from heart failure, and half will die within five years of diagnosis. At least 40% of heart failure patients hve diastolic heart failure (DHF), resulting from slow or incomplete relaxation of the heart muscle in diastole. Calcium (Ca2+) mishandling by cardiac myocytes is an important contributor to DHF. Ca2+ must bind to contractile myofilament proteins in the myocyte to enable contraction in systole, and Ca2+ removal is required for relaxation in diastole. Inefficient Ca2+ removal results in heart stiffening and inability of the ventricles to completely fill with blood for the next heartbeat. The long-term objective of this research is to develop a novel Ca2+ buffering system to facilitate removal of Ca2+ from myofilaments in diastole and restore normal relaxation properties in DHF. Parvalbumin (Parv) is a Ca2+ buffer in fast-twitch muscle, enabling rapid relaxation. Expressing Parv in cardiac cells by viral gene transfer results in rapid relaxation, bu also diminished contraction because Parv pulls Ca2+ away from myofilaments too quickly. Modifying the EF-hand Ca2+ binding domain of Parv (Modified Parv) decreased Ca2+ and increased magnesium (Mg2+) affinities, improving relaxation of cardiac cells while increasing the strength of contraction. In the first aim of this research I will elucidate the mechanism by which Modified Parv increases both relaxation rate and contraction amplitude. The hypothesis is Ca2+ buffering hastens relaxation and Mg2+ buffering amplifies contraction. To test this hypothesis, cells will be isolated from rat hearts, plated on coverslips and treated with virus containing Modified Parv. Contraction/relaxation and Ca2+ cycling will be measured in the presence of Ca2+ channel inhibitors, mutant myofilament proteins with altered Ca2+ sensitivity, and conditions of Ca2+/Mg2+ overload. Contraction/relaxation and Ca2+ cycling will be measured by placing coverslips with cells in a chamber where they are electrically stimulated to contract and visualizing live contractions under a microscope. To measure Ca2+ movement in the cell, a fluorescent dye that binds Ca2+ is used. Faster relaxation and Ca2+ disappearance are expected with Modified Parv.
The second aim will probe the translatability of Modified Parv to improve heart function in two mouse models of DHF. The hypothesis is Modified Parv will improve markers of DHF in these models. To test this hypothesis, Modified Parv transgenic mice will be crossed with cTnI-R193H or Serca2fl/fl;Tg(?MHC-MerCreMer) transgenic mice. Hearts will be excised and cells isolated and analyzed as described above. Whole heart function will also be analyzed. A small water-filled balloon with a pressure transducer will be inserted int the left ventricle of perfused hearts. The heart contracts around the balloon enabling systolic and diastolic pressures to be measured. Decreased diastolic pressure (increased relaxation) is expected from hearts with Modified Parv. Collectively, this experimental plan will enable new insights into Ca2+ mishandling in DHF and establish fundamental knowledge on using Ca2+ buffers to treat the failing heart.
Diastolic heart failure, characterized by slow or incomplete relaxation of the heart, is a growing problem with no cure and a significant socioeconomic burden. A novel calcium buffer for the heart, which improves relaxation rate in diastole while preserving contraction amplitude in systole, has potential to treat or prevent diastolic heart failure. Understanding the mechanisms and clinical applicability of this novel buffer could positively impact length and quality of life for millions of Americans suffering from this deadly disease.
|Wang, Wang; Asp, Michelle L; Guerrero-Serna, Guadalupe et al. (2014) Differential effects of S100 proteins A2 and A6 on cardiac Ca(2+) cycling and contractile performance. J Mol Cell Cardiol 72:117-25|