A common clinical hallmark and characteristic, contributing to the impaired contractile performance in human and experimental heart failure, is the depressed sarcoplasmic reticulum (SR) Ca-cycling. Major emphasis has been placed on correcting either the depressed SR Ca-uptake or impaired SR Ca-release to restore cellular Ca-homeostasis and contractile performance in heart failure. SR Ca-uptake during diastole is mediated by SERCA2a and its reversible regulator phospholamban (PLN);Ca is stored by calsequestrin;and Ca is released during systole through the ryanodine receptor and its anchoring proteins junctin and triadin. Our central hypothesis is that alterations in the fine cross-talk between SR Ca-uptake, storage and release play a critical role in heart failure progression. Our previous studies showed that dephosphorylated phospholamban inhibits the SERCA2 Ca-affinity and it represents a fundamental "brake" mechanism in SR Ca-cycling and cardiac contractility. Indeed, increased phosphorylation of PLN by attenuated protein phosphatase 1 activity through activation of its inhibitor-1 may improve SR Ca-cycling and halt remodeling in the failing heart. Activation of inhibitor-1 occurs by its PKA-phosphorylation at Thr35. However, we have recently shown that cardiac inhibitor-1 has two additional phosphorylation sites: Ser67 and Thr75. Phosphorylation of these two sites increases protein phosphatase 1 activity, resulting in attenuated SR Ca-transport and depressed cardiac contractility. Importantly, phosphorylation of Ser67 and Thr75 is increased in failing hearts. Thus, we propose herein further studies to elucidate the functional role of the newly identified cardiac inhibitor-1 phosphorylation sites by temporal regulation of their expression levels in the adult heart under physiological and stress (pressure-overload and myocardial infarction) conditions. In addition, we uncovered that SR Ca-release may be regulated by junctin and ablation of its expression is associated with delayed after-depolarizations and ventricular arrhythmias. Notably, the junctin levels are almost non-detectable in human failing hearts and this may be a contributing factor to the impaired SR Ca-cycling. Thus, we propose to further elucidate the functional role of junctin in vivo and the mechanisms underlying its regulatory effects on the ryanodine receptor Ca-release channel. We will employ a comprehensive approach integrating studies at the molecular, biochemical and physiological levels. Our preliminary results are very exciting and they support inhibitor-1 and junctin as two key regulators of SR Ca-cycling and nodal points in cardiomyocyte Ca-homeostasis. Overall, the proposed studies will advance our understanding and provide further fundamental insights into the mechanisms underlying regulation of SR Ca-handling in cardiac physiology and pathophysiology.

Public Health Relevance

A universal characteristic of the failing heart 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 (SR) calcium cycling: inhibitor-1, which has been known to regulate protein phosphatase 1 activity and calcium transport into the SR;and junctin, which is present in the sarcoplasmic reticulum lumen and regulates calcium release from the SR. 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 #
5R01HL064018-14
Application #
8429358
Study Section
Special Emphasis Panel (ZRG1-CVRS-K (02))
Program Officer
Lathrop, David A
Project Start
2000-02-01
Project End
2013-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
14
Fiscal Year
2013
Total Cost
$369,923
Indirect Cost
$134,303
Name
University of Cincinnati
Department
Pharmacology
Type
Schools of Medicine
DUNS #
041064767
City
Cincinnati
State
OH
Country
United States
Zip Code
45221
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