Cardiac-specific overexpression of sarcolipin inhibits sarco(endo)plasmic reticulum Ca ²⁺ ATPase (SERCA2a) activity and impairs cardiac function in mice

Abstract

Sarcolipin (SLN) inhibits the cardiac sarco(endo)plasmic reticulum Ca 2؉ ATPase (SERCA2a) by direct binding and is superinhibitory if it binds through phospholamban (PLN). To determine whether overexpression of SLN in the heart might impair cardiac function, transgenic (TG) mice were generated with cardiac-specific overexpression of NF-SLN (SLN tagged at its N terminus with the FLAG epitope). The level of NF-SLN expression (the NF-SLN͞PLN expression ratio) was equivalent to that which induces profound superinhibition when coexpressed with PLN and SERCA2a in HEK-293 cells. In TG hearts, the apparent affinity of SERCA2a for Ca 2؉ was decreased compared with non-TG littermate control hearts. Invasive hemodynamic and echocardiographic analyses revealed impaired cardiac contractility and ventricular hypertrophy in TG mice. Basal PLN phosphorylation was reduced. In isolated papillary muscle subjected to isometric tension, peak amplitudes of Ca 2؉ transients and peak tensions were reduced, whereas decay times of Ca 2؉ transients and relaxation times of tension were increased in TG mice. Isoproterenol largely restored contractility in papillary muscle and stimulated PLN phosphorylation to wild-type levels in intact hearts. No compensatory changes in expression of SERCA2a, PLN, ryanodine receptor, and calsequestrin were observed in TG hearts. Coimmunoprecipitation indicated that overexpressed NF-SLN was bound to both SERCA2a and PLN, forming a ternary complex. These data suggest that NF-SLN overexpression inhibits SERCA2a through stabilization of SERCA2a-PLN interaction in the absence of PLN phosphorylation and through the inhibition of PLN phosphorylation. Inhibition of SERCA2a impairs contractility and calcium cycling, but responsiveness to ␤-adrenergic agonists may prevent progression to heart failure. to the extracellular space. SERCA2a is regulated by phospholamban (PLN) and can potentially be regulated by a homologous protein, sarcolipin (SLN) (1, 2).PLN is a 52-aa SR membrane protein expressed abundantly in cardiac muscle (3). In its dephosphorylated form, PLN interacts with SERCA2a to inhibit Ca 2ϩ transport by lowering the apparent affinity of SERCA2a for Ca 2ϩ . When PLN is phosphorylated, its inhibitory effect on SERCA2a is relieved. The ability of PLN to regulate SERCA2a activity, thereby regulating the rate of cardiac relaxation and the size of the SR Ca 2ϩ store, makes PLN a crucial regulator of cardiac function (1, 2, 4, 5).SLN is a 31-aa SR membrane protein, which has no obvious phosphorylation site (6). Studies based on coexpression of PLN, SLN, and SERCA in heterologous cell culture show that PLN and SLN have similar ability to inhibit either SERCA1a or SERCA2a (7-9). PLN and SLN can form a binary complex that is superinhibitory, presumably because of the formation of a ternary PLN-SLN-SERCA2a complex (9, 10). Thus, SLN can regulate SERCA through either direct interaction with SERCA or through stabilization of the interaction between SERCA and PLN (10).SLN mRNA is expressed abundantly in human fast-twit...

Sarcolipin and phospholamban as regulators of cardiac sarcoplasmic reticulum Ca2+ ATPase

“…SLN overexpression in the ventricle leads to decreased SERCA2a affinity for calcium, Ca 2+ transient amplitude and shortening, and slowed relaxation. Consistent with Asahi et al [46] the +dP/dt and −dP/dt were significantly decreased, due to SLN overexpression. Similar results were found in myocytes and muscle preparations from mice overexpressing SLN, in comparison to the wild-type littermates.…”

“…Overexpression of mouse SLN in the mouse ventricle did not lead to hypertrophy. The development of hypertrophy observed by Asahi et al [46] is probably due to the overexpression of rabbit SLN in the mouse heart, which differs from mouse SLN at the N-terminus. SLN overexpression in the ventricle leads to decreased SERCA2a affinity for calcium, Ca 2+ transient amplitude and shortening, and slowed relaxation.…”

“…Both PLB and SLN inhibit SERCA activity in the heart by lowering the apparent calcium affinity of the pump [42,46,47]. The inhibitory effect of SLN is relieved upon β-adrenergic stimulation, as observed for PLB [13,[46][47][48]. Although, PLB and SLN inhibit SERCA2a activity, there might be subtle but important differences in their mechanism of regulation.…”

“…In the first case, Asahi et al [46] used rabbit cDNA to overexpress SLN in the mouse, by targeting a single copy of the α-MHC driven SLN construct into the Hprt locus of the X-chromosome. This resulted in heterogeneous expression of SLN in female mice due to X-chromosome inactivation.…”

“…The available data suggests that SLN and PLB independently regulate SERCA2a activity [16,20,[46][47][48]. Both PLB and SLN inhibit SERCA activity in the heart by lowering the apparent calcium affinity of the pump [42,46,47].…”