Comparative Proteomics Profiling of a Phospholamban Mutant Mouse Model of Dilated Cardiomyopathy Reveals Progressive Intracellular Stress Responses

Abstract

Defective mobilization of Ca2؉ by cardiomyocytes can lead to cardiac insufficiency, but the causative mechanisms leading to congestive heart failure (HF) remain unclear. In the present study we performed exhaustive global proteomics surveys of cardiac ventricle isolated from a mouse model of cardiomyopathy overexpressing a phospholamban mutant, R9C (PLN-R9C), and exhibiting impaired Ca 2؉ handling and death at 24 weeks and compared them with normal control littermates. The relative expression patterns of 6190 high confidence proteins were monitored by shotgun tandem mass spectrometry at 8, 16, and 24 weeks of disease progression. Significant differential abundance of 593 proteins was detected. These proteins mapped to select biological pathways such as endoplasmic reticulum stress response, cytoskeletal remodeling, and apoptosis and included known biomarkers of HF (e.g. brain natriuretic peptide/atrial natriuretic factor and angiotensin-converting enzyme) and other indicators of presymptomatic functional impairment. These altered proteomic profiles were concordant with cognate mRNA patterns recorded in parallel using high density mRNA microarrays, and top candidates were validated by RT-PCR and Western blotting. Mapping of our highest ranked proteins against a human diseased explant and to available data sets indicated that many of these proteins could serve as markers of disease. Indeed we showed that several of these proteins are detectable in mouse and human plasma and display differential abundance in the plasma of diseased mice and affected patients. These results offer a systems-wide perspective of the dynamic maladaptions associated with impaired Ca

Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function

“…Furthermore, investigation of publicly available cardiac hypertrophy microarray data and protein expression in our published study of a mouse model of dilated cardiomyopathy, based on the R9C point mutation of phospholamban 57 , demonstrated decreased and coordinated expression of both Tmem65 and Cx43 ( Supplementary Fig. 7a,b), suggesting a potential link to cardiac disease.…”

Identification of Novel Ryanodine Receptor 1 (RyR1) Protein Interaction with Calcium Homeostasis Endoplasmic Reticulum Protein (CHERP)

“…Protein Sample Preparation and Proteomic Analysis-Aliquots containing 100 g of total protein from the elution fraction were prepared for trypsin digestion by the addition of ammonium bicarbonate, pH 8.5, to 50 mM and CaCl 2 to a final concentration of 1 mM in a total volume of 180 l. Samples were incubated overnight at 37°C with 0.02 mg/ml proteomics grade trypsin (Roche Applied Science) as described previously (12,13). Before application to the LC-MS columns, the digested peptides were solid-phase extracted using OMIX C-18 pipette tips (Varian Inc.) according to the manufacturer's protocol.…”

“…Comprehensive gel-free shotgun sequencing of tryptic digests was performed essentially as described previously (13,14). Briefly, for peptide separation, individual samples were first loaded onto microcapillary fused silica columns with an internal diameter of 75 m packed with 7 cm of reversed phase C18 resin (Magic C18).…”

Reticulon 4B (Nogo-B) is necessary for macrophage infiltration and tissue repair

“…Previous work has identified Nogo-B as a regulator of vascular remodeling in vivo (7) and cardiac function in mice and humans (8,9). In mice and rabbits, neointimal expansion of injured blood vessels is associated with a marked reduction in endogenous Nogo-B levels suggesting that Nogo-B negatively regulates the extent of vascular injury, and, in humans, the loss of Nogo-B strongly correlates with stenotic lesions and plaque rupture (7,10,11).…”