Rationale Ischemic heart disease is characterized by contractile dysfunction and increased cardiomyocyte death, induced by necrosis and apoptosis. Increased cell survival after an ischemic insult is critical and depends on several cellular pathways, which have not been fully elucidated. Objective To test the hypothesis that the anti-apoptotic haematopoietic-lineage-substrate-1-associated protein-X-1 (HAX-1), recently identified as regulator of cardiac Ca-cycling, may also ameliorate cellular injury in the face of an ischemic insult. Methods and Results We report that cardiac ischemia/reperfusion injury is associated with significant decreases in HAX-1 levels ex vivo and in vivo. Accordingly, over-expression of HAX-1 improved contractile recovery, coupled with reduced infarct size, plasma troponin I level and apoptosis. The beneficial effects were associated with decreased ER stress response through specific inhibition of the inositol-requiring-enzyme (IRE-1) signaling pathway, including its downstream effectors caspase-12 and the transcription factor C/EBP homologous protein. Conversely, HAX-1 heterozygous deficient hearts exhibited increases in infarct size and IRE-1 activity. The inhibitory effects of HAX-1 were mediated by its binding to the N-terminal fragment of the heat shock protein 90 (Hsp90). Moreover, HAX-1 sequestered Hsp90 from IRE-1 to the phospholamban/SERCA calcium transport complex. The HAX-1 regulation was further supported by loss of IRE-1 inhibition in presence of the Hsp90 inhibitor, 17-N-Allylamino-17-Demethoxygeldanamycin. Conclusions Cardiac ischemia/reperfusion injury is associated with decreases in HAX-1 levels. Consequently, over-expression of HAX-1 promotes cardiomyocyte survival, mediated by its interaction with Hsp90 and specific inhibition of IRE-1 signaling at the ER/SR.
RATIONALE Heat shock proteins (Hsps) are known to enhance cell survival under various stress conditions. In the heart, the small Hsp20 has emerged as a key mediator of protection against apoptosis, remodeling and ischemia/reperfusion injury. Moreover, Hsp20 has been implicated in modulation of cardiac contractility ex vivo. OBJECTIVE To determine the in vivo role of Hsp20 in the heart and the mechanisms underlying its regulatory effects in Ca-cycling. METHODS and RESULTS Hsp20 overexpression resulted in significant enhancement of cardiac function in intact animals, coupled with augmented Ca-cycling and sarcoplasmic reticulum (SR) Ca-load in isolated cardiomyocytes. This was associated with specific increases in phosphorylation of phospholamban (PLN) at both Ser16 and Thr17, relieving its inhibition of the apparent Ca-affinity of SERCA2a. Accordingly, the inotropic effects of Hsp20 were abrogated in cardiomyocytes expressing non-phosphorylatable PLN (S16A/T17A). Interestingly, the activity of type 1 protein phosphatase (PP1), a known regulator of PLN signaling, was significantly reduced by Hsp20-overexpression, suggesting that the Hsp20 stimulatory effects are partially mediated through the PP1/PLN axis. This hypothesis was supported by cell-fractionation, co-immunoprecipitation and co-immunolocalization studies, which revealed an association between Hsp20, PP1 and PLN. Furthermore, recombinant protein studies confirmed a physical interaction between AA 73–160 in Hsp20 and AA 163–330 in PP1. CONCLUSIONS Hsp20 is a novel regulator of SR Ca-cycling by targeting the PP1/PLN axis. These findings, coupled with the well-recognized cardioprotective role of Hsp20, suggest a dual benefit of targeting Hsp20 in heart disease.
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