The Role of Small Heat Shock Protein 20 and Its Phosphorylation in the Regulation of Cardiac Function and Ischemia/Reperfusion Injury

The small heat shock protein (sHsp) with apparent molecular mass of 20 kD (Hsp20) is one of 10 members of the sHsp family. Interestingly, Hsp20 is the only member within this family that contains a consensus peptide motif (RRAS) for protein kinase A (PKA)/protein kinase G (PKG)-dependent phosphorylation at Ser16. Recent studies have shown that enhanced myocardial function was associated with increased expression levels of Hsp20 and its phosphorylation. To further elucidate the possible mechanisms underlying the inotropic effects of Hsp20 and its phosphorylation, as well as their possible roles in ischemia/reperfusion-induced cardiac injury, the present study employed in vitro adenoviral-gene transfer and in vivo transgenic approaches.

Our study firstly revealed that acute overexpression of wild-type Hsp20 by adenoviral infection augmented cardiac myocyte contractility, which was further confirmed in Hsp20-transgenic murine hearts (10-fold overexpression). This hypercontractility was associated with increased activation of phospholamban (PLN), evidenced by ≈ 2-fold higher expression of Ser16/Thr17-phosphorylated PLN in Hsp20-transgenic hearts related to non-transgenic controls. Furthermore, co-immunoprecipitation experiments indicated that Hsp20 was associated with type 1 phosphatase (PP1), suggesting Hsp20 may regulate PP1 activity in the mouse heart. Indeed, PP1 activity was significantly reduced in Hsp20-transgenic hearts, compared to non-transgenic hearts. These results imply that Hsp20 positively regulate cardiac function via inhibition of PP1 activity, and its downstream target, PLN phosphorylation.

Secondly, to further assess the functional significance of p-Ser16 Hsp20 in vivo and its possible roles in regulation of I/R-induced apoptosis and autophagy, we generated a transgenic mouse model with cardiac-specific expression of a non-phosphorylatable Hsp20 (Hsp20^S16A). Our findings indicate that increased Hsp20^S16A expression in the heart failed to protect hearts against ex vivo and in vivo I/R injury, accompanied by impaired autophagy and increased apoptosis. Accordingly, pre-treatment of Hsp20^S16A hearts with rapamycin, an activator of autophagy, resulted in improvement of functional recovery, compared with saline-treated Hsp20^S16A hearts. Thus, Hsp20 and its Ser16 phosphorylation may be involved in the regulation of I/R-induced cardiac autophagy and cell death.

Finally, we generated the Hsp20^S16D transgenic mouse model, in which Ser16 is replaced with aspartic acid (D), to further explore the in vivo role of p-Ser16 Hsp20. Surprisingly, we observed that contractile parameters were significantly depressed in Hsp20^S16D cardiomyocytes. In vivo contractile function was also significantly impaired in TGs, compared with their non-transgenic littermates. In addition, TG mice developed left ventricular (LV) fibrosis at 6 weeks of age, without evidence of LV hypertrophy or dilation, and their life span was markedly shortened (mean age at death: 9 months). Further electron microscopy examination of the hearts revealed that double-membrane autophagosomes were more prominent in TGs. This was associated with increased lysosomal activity, suggesting that autophagosome accumulation was not due to diminished activity of distal lysosomal pathways. Our data indicate that long-term augmentation of cardiac Hsp20 phosphorylation impairs cardiac function, accentuates pathological remodeling and increases autophagic activity, leading to premature death.

Collectively, these studies demonstrate that Hsp20 may be a key regulator of Ca2+-cycling through modulation of PP1-PLN activity, and phosphorylation of Hsp20 is important in the regulation of ischemia/reperfusion-induced cardiac autophagy and cell death.