The overall goal of this proposal is to understand calcium regulation in both cardiac and skeletal muscle at the atomic level. Calcium (Ca2+) is an essential messenger for muscle contractility, and its homeostatic balance is controlled by proteins embedded or peripheral to the sarcoplasmic reticulum (SR) membrane. In cardiac myocytes, the SR Ca2+-ATPase (SERCA) regulates diastole by translocating ~70% of the Ca2+ ions. In its unphosphorylated state, phospholamban (PLN) reduces SERCA's affinity for Ca2+, whereas phosphorylation of PLN at Ser16 re-establishes basal Ca2+ transport. Sarcolipin (SLN), a PLN homolog, regulates SERCA in both skeletal and cardiac muscle, keeping SERCA's activity within a physiological window. Like PLN, SLN is also modulated by ?-adrenergic stimulation through phosphorylation at Thr5, though the role of this phosphory- lation event in SERCA regulation is incompletely understood. Dysregulation of SERCA's function outside the physiological window by PLN and SLN leads to cardiomyopathies and heart failure. SLN is also proposed to play a crucial role in metabolism and skeletal muscle thermogenesis through uncoupling ATP hydrolysis and Ca2+ transport in SERCA. Though there is compelling biological evidence for this phenomenon, the molecular mechanisms by which SLN elicit the functional uncoupling of SERCA is unknown. In this competitive renewal, we will build on our previous discoveries and take new and exciting directions to understand the atomic details of PLN and SLN's allosteric regulation of SERCA and unveil the different roles of these endogenous inhibi- tors both in cardiac and skeletal muscle.
In AIM1, We will focus on the structural analysis of SERCA regulation by two mutants, PLNP21G and PLNM20GP21G, which we designed and currently testing for gene therapy. We will study how these mutants mimic the phosphorylated state of PLN and tune SERCA activity.
In AIM2, we will determine the role of SLN in non-shivering muscle thermogenesis. Finally, in AIM3, we will study the functional and structural role of a newly discovered regulator of the SERCA/PLN complex: HAX-1, which has an anti- apoptotic role in cardiomyocytes. These studies will be carried out using a combination of molecular biology, biochemical assays, thermocalorimetry, and spectroscopic methods (solution, solid-state NMR and fluores- cence) in synthetic and native membranes. To accomplish these specific AIMs, we have assembled a strong collaborative team, including Dave Thomas, Muthu Periasamy, Roger Hajjar, Evangelia Kranias, and Seth Robia. These scientists are at the forefront of the study of Ca2+ physiology and pathophysiology and have committed to a collaborative effort and timely data sharing plan that will propel our research and ensure sound biological and biomedical significance for our studies. The outcomes of this research will have a strong im- pact for understanding excitation-contraction coupling, muscle contractility, and thermogenesis, with significant implications for the design of alternative therapeutic approaches to counteract muscle disease.

Public Health Relevance

Finding therapeutic strategies to effectively tune Ca2+ cycling in response to the different mani- festations of heart disease remains a `holy grail.' We propose to study the allosteric interactions between the sarcoplasmic reticulum Ca2+-ATPase and three regulatory proteins (phosphol- amban, sarcolipin, and HAX-1). Since dysregulation of SERCA is directly linked to cardiac and skeletal muscle diseases, such as hypertrophic and dilated cardiomyopathies, Brody's disease, and Duchenne muscular dystrophy, understanding the structural details of these interactions is central to designing innovative therapies to treat these devastating diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM064742-17
Application #
9689316
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Mcguirl, Michele
Project Start
2002-09-16
Project End
2021-04-30
Budget Start
2019-05-01
Budget End
2021-04-30
Support Year
17
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Biochemistry
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Chipot, Christophe; Dehez, François; Schnell, Jason R et al. (2018) Perturbations of Native Membrane Protein Structure in Alkyl Phosphocholine Detergents: A Critical Assessment of NMR and Biophysical Studies. Chem Rev 118:3559-3607
Nelson, Sarah E D; Ha, Kim N; Gopinath, Tata et al. (2018) Effects of the Arg9Cys and Arg25Cys mutations on phospholamban's conformational equilibrium in membrane bilayers. Biochim Biophys Acta Biomembr 1860:1335-1341
Wang, Songlin; Gopinath, T; Veglia, Gianluigi (2018) Application of paramagnetic relaxation enhancements to accelerate the acquisition of 2D and 3D solid-state NMR spectra of oriented membrane proteins. Methods 138-139:54-61
Gopinath, T; Nelson, Sarah E D; Veglia, Gianluigi (2017) 1H-detected MAS solid-state NMR experiments enable the simultaneous mapping of rigid and dynamic domains of membrane proteins. J Magn Reson 285:101-107
Harmouche, Nicole; Aisenbrey, Christopher; Porcelli, Fernando et al. (2017) Solution and Solid-State Nuclear Magnetic Resonance Structural Investigations of the Antimicrobial Designer Peptide GL13K in Membranes. Biochemistry 56:4269-4278
Manu, V S; Veglia, Gianluigi (2016) Optimization of identity operation in NMR spectroscopy via genetic algorithm: Application to the TEDOR experiment. J Magn Reson 273:40-46
Gopinath, T; Veglia, Gianluigi (2016) Multiple acquisitions via sequential transfer of orphan spin polarization (MAeSTOSO): How far can we push residual spin polarization in solid-state NMR? J Magn Reson 267:1-8
Sanz-Hernández, Máximo; Vostrikov, Vitaly V; Veglia, Gianluigi et al. (2016) Accurate Determination of Conformational Transitions in Oligomeric Membrane Proteins. Sci Rep 6:23063
Dicke, Alysha; Gopinath, Tata; Wang, Yingjie et al. (2016) Probing Residue-Specific Water-Protein Interactions in Oriented Lipid Membranes via Solid-State NMR Spectroscopy. J Phys Chem B :
Vostrikov, Vitaly V; Gustavsson, Martin; Gopinath, Tata et al. (2016) Ca(2+) ATPase Conformational Transitions in Lipid Bilayers Mapped by Site-directed Ethylation and Solid-State NMR. ACS Chem Biol 11:329-34

Showing the most recent 10 out of 80 publications