Human dilated cardiomyopathy (DCM) is associated with increased Gi protein levels, increased Gi signaling, and auto-antibodies that activate signaling by GI- coupled receptors. The goal of this proposal is to test the hypothesis that Gi signaling can cause DCM. Control of Gi signaling has been achieved by expressing in the heart a Gi-coupled receptor that has been specifically designed to be a Receptor Activated Solely by a Synthetic Ligand, or RASSL. The first RASSL (R1) is based on a Gi-coupled, kappa- opioid receptor. R1 contains mutations that reduce affinity for natural peptide agonists and yet allow activation by the drug spiradoline. Cardiac-specific, conditional expression of R1 in transgenic mice is achieved with a tetracycline-controlled expression system utilizing the a-myosin heavy chain promoter. Activation of R1 signaling by spiradoline administration results in acute slowing of heart rate and complete atrioventricular block, which are known effects of Gi signaling. Preliminary studies show that prolonged signaling by R1 causes a lethal form of congestive heart failure with anasarca (up to 60% weight gain), contractile dysfunction, and the histopathological features of DCM. The Gi signaling-induced cardiomyopathy can be phenotypically reversed by suppressing R1 expression, creating the potential for studies of disease recovery as well as disease onset.
Specific Aims are: (1) to determine if receptor- stimulated Gi signaling in the heart can cause the characteristic anatomical, physiological and histopathological changes of DCM using echocardiography, perfused hearts, isolated heart tissue strips, quantitative morphometric analysis, gene expression, and biochemical markers of cardiomyopathy; (2) to determine if the DCM is influenced by the spatial or temporal nature of the Gi signal, by altering the anatomical location of the Gi signal, inducing continuous Gi signaling with a mutationally activated form of Gi, and reducing continuous basal Gi signaling by expressing a new RASSL (R2) that has a lower susceptibility to endogenous peptide agonists; and (3) to determine if a mouse heart with Gi-induced DCM can regain normal function on a physiologic, histologic, and cellular level.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL060664-02
Application #
6030901
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Project Start
1998-07-01
Project End
2003-06-30
Budget Start
1999-07-01
Budget End
2000-06-30
Support Year
2
Fiscal Year
1999
Total Cost
Indirect Cost
Name
J. David Gladstone Institutes
Department
Type
DUNS #
047120084
City
San Francisco
State
CA
Country
United States
Zip Code
94158
Miyaoka, Yuichiro; Mayerl, Steven J; Chan, Amanda H et al. (2018) Detection and Quantification of HDR and NHEJ Induced by Genome Editing at Endogenous Gene Loci Using Droplet Digital PCR. Methods Mol Biol 1768:349-362
Hayashi, Yohei; Hsiao, Edward C; Sami, Salma et al. (2016) BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence. Proc Natl Acad Sci U S A 113:13057-13062
Huang, Miller; Miller, Matthew L; McHenry, Lauren K et al. (2016) Generating trunk neural crest from human pluripotent stem cells. Sci Rep 6:19727
Mandegar, Mohammad A; Huebsch, Nathaniel; Frolov, Ekaterina B et al. (2016) CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs. Cell Stem Cell 18:541-53
Miyaoka, Yuichiro; Berman, Jennifer R; Cooper, Samantha B et al. (2016) Systematic quantification of HDR and NHEJ reveals effects of locus, nuclease, and cell type on genome-editing. Sci Rep 6:23549
Kime, Cody; Mandegar, Mohammad A; Srivastava, Deepak et al. (2016) Efficient CRISPR/Cas9-Based Genome Engineering in Human Pluripotent Stem Cells. Curr Protoc Hum Genet 88:Unit 21.4
Orr, Anna G; Hsiao, Edward C; Wang, Max M et al. (2015) Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory. Nat Neurosci 18:423-34
Wang, Liping; Hsiao, Edward C; Lieu, Shirley et al. (2015) Loss of Gi G-Protein-Coupled Receptor Signaling in Osteoblasts Accelerates Bone Fracture Healing. J Bone Miner Res 30:1896-904
Spencer, C Ian; Baba, Shiro; Nakamura, Kenta et al. (2014) Calcium transients closely reflect prolonged action potentials in iPSC models of inherited cardiac arrhythmia. Stem Cell Reports 3:269-81
Park, Jason S; Rhau, Benjamin; Hermann, Aynur et al. (2014) Synthetic control of mammalian-cell motility by engineering chemotaxis to an orthogonal bioinert chemical signal. Proc Natl Acad Sci U S A 111:5896-901

Showing the most recent 10 out of 32 publications