Specialized compartments of cardiac nuclei exhibit distinct proteomic anatomy.

As host to the genome, the nucleus plays a critical role as modulator of cellular phenotype. To understand the totality of proteins that regulate this organelle, we used proteomics to characterize the components of the cardiac nucleus. Following purification, cardiac nuclei were fractionated into biologically relevant fractions including acid-soluble proteins, chromatin-bound molecules and nucleoplasmic proteins. These distinct subproteomes were characterized by liquid chromatography-tandem MS. We report a cardiac nuclear proteome of 1048 proteins--only 146 of which are shared between the distinct subcompartments of this organelle. Analysis of genomic loci encoding these molecules gives insights into local hotspots for nuclear protein regulation. High mass accuracy and complementary analytical techniques allowed the discrimination of distinct protein isoforms, including 54 total histone variants, 17 of which were distinguished by unique peptide sequences and four of which have never been detected at the protein level. These studies are the first unbiased analysis of cardiac nuclear subcompartments and provide a foundation for exploration of this organelle's proteomes during disease.

Stress signaling by Tec tyrosine kinase in the ischemic myocardium.

Nonreceptor tyrosine kinases have an increasingly appreciated role in cardiac injury and protection. To investigate novel tasks for members of the Tec family of nonreceptor tyrosine kinases in cardiac phenotype, we examined the behavior of the Tec isoform in myocardial ischemic injury. Ischemia-reperfusion, but not cardiac protective agents, induced altered intracellular localization of Tec, highlighting distinct actions of this protein compared with other isoforms, such as Bmx, in the same model. Tec is abundantly expressed in cardiac myocytes and assumes a diffuse intracellular localization under basal conditions but is recruited to striated structures upon various stimuli, including ATP. To characterize Tec signaling targets in vivo, we performed an exhaustive proteomic analysis of Tec-binding partners. These experiments expand the role of the Tec family in the heart, identifying the Tec isoform as an ischemic injury-induced isoform, and map the subproteome of its interactors in isolated cells.

Post-translational regulation of calsarcin-1 during pressure overload-induced cardiac hypertrophy.

Chronic pressure overload to the heart leads to cardiac hypertrophy and failure through processes that involve reorganization of subcellular compartments and alteration of established signaling mechanisms. To identify proteins contributing to this process, we examined changes in nuclear-associated myofilament proteins as the murine heart undergoes progressive hypertrophy following pressure overload. Calsarcin-1, a negative regulator of calcineurin signaling in the heart, was found to be enriched in cardiac nuclei and displays increased abundance following pressure overload through a mechanism that is decoupled from transcriptional regulation. Using proteomics, we identified novel processing of this protein in the setting of cardiac injury and identified four residues subject to modification by phosphorylation. These studies are the first to determine mechanisms regulating calsarcin abundance during hypertrophy and failure and reveal the first evidence of post-translational modifications of calsarcin-1 in the myocardium. Overall, the findings expand the roles of calsarcins to include nuclear tasks during cardiac growth.

Loss of Bmx nonreceptor tyrosine kinase prevents pressure overload-induced cardiac hypertrophy.

Bmx nonreceptor tyrosine kinase has an established role in endothelial and lymphocyte signaling; however, its role in the heart is unknown. To determine whether Bmx participates in cardiac growth, we subjected mice deficient in the molecule (Bmx knockout mice) to transverse aortic constriction (TAC). In comparison with wild-type mice, which progressively developed massive hypertrophy following TAC, Bmx knockout mice were resistant to TAC-induced cardiac growth at the organ and cell level. Loss of Bmx preserved cardiac ejection fraction and decreased mortality following TAC. These findings are the first to demonstrate a necessary role for the Tec family of tyrosine kinases in the heart and reveal a novel regulator (Bmx) of pressure overload-induced hypertrophic growth.