Novel mechanisms of phospholamban/SERCA2a modulation: Phosphorylation vs S-nitrosylation and S-sulfydration

Phospholamban (PLN), a small protein closely associated with the cardiac sarcoplasmic reticulum (SR), has been identified and sequenced in many vertebrates, from fish to human. The very high homology among the sequence underlines the old evolutionary history of this protein, as well as its importance in myocytes dynamics. By modulating the intracellular calcium transient it represents the major determinant of cardiac contractility and relaxation. Alternate PLN phosphorylation/dephosphorylation determines SERCA2a on/off state, and thus the rate of SR refilling with Ca2+, with a consequent impact on myocardial relaxation and contraction. In its dephosphorylated state, PLN inhibits Ca2+ sequestration by SERCA2a (1) and this induces more Ca2+ to be available for the contractile apparatus, thus decelerating relaxation; when PLN is phosphorylated this inhibition is relieved and Ca2+ is actively pumped into the SR causing an increased rate of myocardial relaxation (2). A phosphorylation-dependent activation of PLN was observed by us both in mammalian (rat: 3, 4, 5) and non-mammalian (fish: 6, amphibian: 7) vertebrate hearts. Recently, we have identified alternative, phosphorylation-independent, mechanisms of PLN/SERCA2a regulated Ca2+ reuptake, such as S-nitrosylation (6, 4, 5) and S-sulfhydration (8).S-nitrosylation, the covalent modification of a protein cysteine thiol by a nitric oxide (NO) group to generate an S-nitrosothiol (SNO), is recognized to be important in regulating protein function (9). This is particularly relevant in the heart, in which several proteins of critical significance were identified as potential targets for S-nitrosylation (10). Using the biotin switch method (11) (Fig. 1), we demonstrated that in the eel (6) and rat (4, 5) heart PLN represents an important target for S-nitrosylation. In particular, in the eel it has been observed that the Frank-Starling response is modulated by a nitric oxide-dependent S-nitrosylation of PLN, which in turn improves myocardial relaxation (6). In the rat heart, PLN S-nitrosylation appeared involved in the lusitropic action of several cardioactive substances, such as Catestatin and 17–β-estradiol (4,5).A modified biotin switch method, using S-methyl methanethiosulfonate (MMTS) as an alkylating reagent, was recently used to identify a large number of proteins that may undergo S-sulfhydration (12). Similarly to S-nitrosylation, S-sulfhydration represents an important signal which modulates many biological processes. In frog and rat heart, NaHS (a donor of H2S) increases protein S-sulfhydration (8). In the rat, Western Blotting of membrane fraction revealed two bands corresponding to the apparent molecular weights of PLN monomer (6kDa) and dimer (12 kDa) as putative targets for S-sulfhydration. This was confirmed by immunoprecipitation of the membrane protein fraction with anti-PLN antibody which revealed an increase of PLN S-sulfhydration in NaHS-treated hearts, particularly evident in the case of the 12kDa band (8) (Fig. 2).Taken together, these results propose S-nytrosilation and S-sulfhydration of PLN as novel mechanisms for SERCA2a regulation which in turn modulates myocardial inotropic and lusitropic properties. References1) Kimura Y, Kurzydlowski K, Tada M, MacLennan DH. Phospholamban inhibitory function is activated by depolymerization. J Biol Chem 272: 15061–15064, 1997.2) Schmidt AG, Edes I, Kranias EG. Phospholamban: a promising therapeutic target in heart failure? Cardiovasc Drugs Ther 15: 387–396, 2001.3) Angelone T, Quintieri A M, Brar BK, Limchiyawat PT, Tota B, Mahata SK, Cerra MC. The antihypertensive chromogranin a peptide catestatin acts as a novel endocrine/paracrine modulator of cardiac inotropism and lusitropism. 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