A key characteristic of human and experimental heart failure is impaired calcium cycling through the sarcoplasmic reticulum (SR), which contributes to deteriorated contractility. The major functions of the SR are: Ca-uptake mediated by SERCA2a and its reversible regulator phospholamban (PLN);Ca-storage through calsequestrin and the histidine rich Ca-binding protein (HRC);and Ca-release occurring through the ryanodine receptor complex. Our central hypothesis is that alterations in the fine-tuned regulation of SR Ca-cycling play a critical role in heart failure progression. The long-term goal of this project is to determine the triggers and mechanisms, which disrupt SR function, to reveal new therapeutic targets. Our previous studies showed that phospholamban interacts with SERCA2 and inhibits its Ca-affinity, representing a fundamental """"""""brake"""""""" in SR Ca-cycling and cardiac contractility. Thus, inhibition of PLN activity to normalize the depressed Ca- homeostasis has been suggested to be of therapeutic benefit in failing hearts. However, we have recently shown that phospholamban also interacts with the anti-apoptotic HS-1 associated protein X-1 (HAX-1). Actually, HAX-1 enhances the inhibitory effects of phospholamban on SERCA2a, while isoproterenol stimulation relieves this inhibition. In addition, we uncovered that HRC interacts with SERCA2a and regulates the maximal rates of Ca-transport. Thus, we have identified a multimeric complex (HAX- 1/PLN/SERCA2a/HRC), which mediates SR Ca-uptake in the heart. However, the functional significance of these newly identified regulators of SR Ca-cycling is not currently clear. We propose here to: a) define the role of HAX-1 by temporal regulation of its expression levels in the adult heart to assess its function on SR Ca- cycling and cardiomyocyte apoptosis under physiological and stress conditions;and b) elucidate the functional significance of HRC ablation and the human S96A-HRC variant in cardiac SR Ca-uptake and release as well as overall cardiomyocyte function. We will employ an integrated approach with studies at the molecular, biochemical and physiological levels. Our preliminary results in each of the specific aims make the proposed studies attractive and feasible, especially since changes in the levels or activity of Hax-1 and HRC are expected to be linked to changes in cardiac function and/or cell death. These studies will provide further fundamental insights into the role of SR Ca-handling in cardiac physiology and pathophysiology.

Public Health Relevance

A universal characteristic of the failing hearts is depressed calcium cycling through the sarcoplasmic reticulum, which reflects deteriorated heart function. This proposal concentrates on elucidating the role of two proteins involved in sarcoplasmic reticulum calcium cycling: HAX-1, which has been known to regulate cell survival;and the histidine rich calcium binding protein (HRC), which is present in the sarcoplasmic reticulum lumen. Our studies will clearly advance our knowledge on these two protein players involved in cardiac function and dysfunction, which may lead to better therapeutic avenues in heart failure.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL026057-32
Application #
8300836
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Krull, Holly
Project Start
1981-07-01
Project End
2013-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
32
Fiscal Year
2012
Total Cost
$386,100
Indirect Cost
$138,600
Name
University of Cincinnati
Department
Pharmacology
Type
Schools of Medicine
DUNS #
041064767
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
Mazzocchi, G; Sommese, L; Palomeque, J et al. (2016) Phospholamban ablation rescues the enhanced propensity to arrhythmias of mice with CaMKII-constitutive phosphorylation of RyR2 at site S2814. J Physiol 594:3005-30
Bidwell, Philip A; Kranias, Evangelia G (2016) Calcium Uptake in Crude Tissue Preparation. Methods Mol Biol 1377:161-70
Tzimas, Christos; Terrovitis, John; Lehnart, Stephan E et al. (2015) Calcium/calmodulin-dependent protein kinase II (CaMKII) inhibition ameliorates arrhythmias elicited by junctin ablation under stress conditions. Heart Rhythm 12:1599-610
Liu, Bin; Ho, Hsiang-Ting; Brunello, Lucia et al. (2015) Ablation of HRC alleviates cardiac arrhythmia and improves abnormal Ca handling in CASQ2 knockout mice prone to CPVT. Cardiovasc Res 108:299-311
Bibli, Sofia-Iris; Andreadou, Ioanna; Chatzianastasiou, Athanasia et al. (2015) Cardioprotection by H2S engages a cGMP-dependent protein kinase G/phospholamban pathway. Cardiovasc Res 106:432-42
Cai, Wen-Feng; Liu, Guan-Sheng; Lam, Chi Keung et al. (2015) Up-regulation of micro-RNA765 in human failing hearts is associated with post-transcriptional regulation of protein phosphatase inhibitor-1 and depressed contractility. Eur J Heart Fail 17:782-93
Haghighi, Kobra; Pritchard, Tracy J; Liu, Guan-Sheng et al. (2015) Human G109E-inhibitor-1 impairs cardiac function and promotes arrhythmias. J Mol Cell Cardiol 89:349-59
Karakikes, Ioannis; Stillitano, Francesca; Nonnenmacher, Mathieu et al. (2015) Correction of human phospholamban R14del mutation associated with cardiomyopathy using targeted nucleases and combination therapy. Nat Commun 6:6955
Lam, Chi Keung; Zhao, Wen; Liu, Guan-Sheng et al. (2015) HAX-1 regulates cyclophilin-D levels and mitochondria permeability transition pore in the heart. Proc Natl Acad Sci U S A 112:E6466-75
Irie, Tomoya; Sips, Patrick Y; Kai, Shinichi et al. (2015) S-Nitrosylation of Calcium-Handling Proteins in Cardiac Adrenergic Signaling and Hypertrophy. Circ Res 117:793-803

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