Abstract 2944: Activation of Heat Shock Proteins and Shifts in Cytoskeleton Composition in a Cardiac-specific SERCA2 KO Mouse
BACKGROUND: Depressed contractility is a key feature of the failing heart and has been linked to reduced Ca2+ availability due to decreased activity of the sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA2). Yet, surprisingly, a conditional, cardiac specific SERCA 2 knock out (KO) mouse, has sustained cardiac contractility for several weeks despite dramatically altered cytosolic Ca2+ handling and SR function. The molecular mechanism behind this intriguing contractile compensation is not clear.
AIM: To examine the protein alterations in the cytosolic sub-proteome and identify the biological processes altered in the KO myocardium that can compensate for the loss of SERCA2 in the heart.
METHODS AND RESULTS: SERCA2 KO and age matched control mice analyzed 9 days after induced gene excision showed no differences in cardiac function (left atrial diameter, echocardiography), even though the quantity of SERCA2 protein was reduced to 30±5% in the left ventricles in KO vs. control mice (western blot). The cytosolic-enriched protein extracts from KO and control hearts (n=6) was analyzed by two dimensional gel electrophoresis (pH 4–7 and 6–11) and 33 cytosolic proteins were identified as being altered (Redfin, Ludesi). The majority of the proteins were identified (tandem mass spectrometry, MS2) and analyzed further using seeded Bayesian networks. The main changes occurred to the cytoskeleton composition with alterations in vinculin, actin, gelsolin and coffilin-2. These were accompanied by regulation of a subset of heat shock proteins (HSPs), HSPB1, alpha-B-crystallin and HSPA5. Further analysis of the HSPs (Immobilized metal affinity chromatography and MS2) showed an increased level of the phosphorylated (activated) forms. Finally, these results were compared to effects in acute thapsigargin-treated neonatal cardiomyocytes to examine whether the observed proteomic changes in SERCA2 KO hearts occur in vivo as a direct consequence of altered Ca2+ homeostasis.
CONCLUSIONS AND PERSPECTIVES: Altered Ca2+ homeostasis induces activation of HSPs and shifts in cytoskel-eton protein composition. This indicates that Ca2+ changes can induce phenotypical effects beyond the contractile apparatus, also involving the architecture of the cardiomyocytes.