Cardiac calsequestrin (Casq2) is thought to be the key sarcoplasmic reticulum (SR) Ca 2+ storage protein essential for SR Ca 2+ release in mammalian heart. Human CASQ2 mutations are associated with catecholaminergic ventricular tachycardia. However, homozygous mutation carriers presumably lacking functional Casq2 display surprisingly normal cardiac contractility. Here we show that Casq2-null mice are viable and display normal SR Ca 2+ release and contractile function under basal conditions. The mice exhibited striking increases in SR volume and near absence of the Casq2-binding proteins triadin-1 and junctin; upregulation of other Ca 2+ -binding proteins was not apparent. Exposure to catecholamines in Casq2-null myocytes caused increased diastolic SR Ca 2+ leak, resulting in premature spontaneous SR Ca 2+ releases and triggered beats. In vivo, Casq2-null mice phenocopied the human arrhythmias. Thus, while the unique molecular and anatomic adaptive response to Casq2 deletion maintains functional SR Ca 2+ storage, lack of Casq2 also causes increased diastolic SR Ca 2+ leak, rendering Casq2-null mice susceptible to catecholaminergic ventricular arrhythmias.

Heart muscle excitation-contraction (E-C) coupling is governed by Ca 2؉ release units (CRUs) whereby Ca 2؉ influx via L-type Ca 2؉ channels (Cav1.2) triggers Ca 2؉ release from juxtaposed Ca 2؉ release channels (RyR2) located in junctional sarcoplasmic reticulum (jSR). Although studies suggest that the jSR protein triadin anchors cardiac calsequestrin (Casq2) to RyR2, its contribution to E-C coupling remains unclear. Here, we identify the role of triadin using mice with ablation of the Trdn gene (Trdn ؊/؊ ). The structure and protein composition of the cardiac CRU is significantly altered in Trdn ؊/؊ hearts. jSR proteins (RyR2, Casq2, junctin, and junctophilin 1 and 2) are significantly reduced in Trdn ؊/؊ hearts, whereas Cav1.2 and SERCA2a remain unchanged. Electron microscopy shows fragmentation and an overall 50% reduction in the contacts between jSR and T-tubules. Immunolabeling experiments show reduced colocalization of Cav1.2 with RyR2 and substantial Casq2 labeling outside of the jSR in Trdn ؊/؊ myocytes. CRU function is impaired in Trdn ؊/؊ myocytes, with reduced SR Ca 2؉ release and impaired negative feedback of SR Ca 2؉ release on Cav1.2 Ca 2؉ currents (ICa). Uninhibited Ca 2؉ influx via ICa likely contributes to Ca 2؉ overload and results in spontaneous SR Ca 2؉ releases upon ␤-adrenergic receptor stimulation with isoproterenol in Trdn ؊/؊ myocytes, and ventricular arrhythmias in Trdn ؊/؊ mice. We conclude that triadin is critically important for maintaining the structural and functional integrity of the cardiac CRU; triadin loss and the resulting alterations in CRU structure and protein composition impairs E-C coupling and renders hearts susceptible to ventricular arrhythmias.cardiac muscle ͉ sarcoplasmic reticulum ͉ calsequestrin ͉ Cav1.2 ͉ RyR2

Background: Phospholamban (PLB) regulates sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA) activity and is thus a key regulator of cardiac contractility. Results: We present the crystal structure of SERCA in complex with PLB at 2.8-Å resolution. Conclusion: PLB stabilizes a divalent cation-free conformation of SERCA with collapsed Ca 2ϩ binding sites. We call the structure E2-PLB. Significance: The E2-PLB structure explains how PLB decreases Ca 2ϩ affinity and depresses cardiac contractility.

Interactions between the transmembrane domains of phospholamban (PLB) and the cardiac Ca 2؉ pump (SERCA2a) have been investigated by chemical cross-linking. Specifically, C-terminal, transmembrane residues 45-52 of PLB were individually mutated to Cys, then cross-linked to V89C in the M2 helix of SERCA2a with the thiol-specific cross-linking reagents Cu 2؉ -phenanthroline, dibromobimane, and bismaleimidohexane. V49C-, M50C-, and L52C-PLB all crosslinked strongly to V89C-SERCA2a, coupling to 70 -100% of SERCA2a molecules. Residues 45-48 and 51 of PLB also cross-linked to V89C of SERCA2a, but more weakly. Evidence for the mechanism of PLB regulation of SERCA2a was provided by the conformational dependence of cross-linking. In particular, the required absence of Ca 2؉ for crosslinking implicated the E2 conformation of SERCA2a, and its enhancement by ATP confirmed E2⅐ATP as the conformation with the highest affinity for PLB. In contrast, E2 phosphorylated with inorganic phosphate (E2P) and E2 inhibited by thapsigargin (E2⅐TG) both failed to cross-link to PLB. These results with transmembrane PLB residues are completely consistent with cytoplasmic PLB residues studied previously, suggesting that the dissociation of PLB from the Ca 2؉ pump is complete, not partial, when the pump binds Ca 2؉ (E1⅐Ca 2 ) or adopts the E2P or E2⅐TG conformations. V49C of PLB cross-linked to 100% of SERCA2a molecules, suggesting that this residue might have functional importance for regulation. Indeed, we found that mutation of Val 49 to smaller side-chained residues V49A or V49G augmented PLB inhibition, whereas mutation to the larger hydrophobic residue, V49L, prevented PLB inhibition. A model for the interaction of PLB with SERCA2a is presented, showing that Val 49 fits into a constriction at the lumenal end of the M2 helix of SERCA, possibly controlling access of PLB to its binding site on SERCA. Phospholamban (PLB)2 is a small membrane protein of only 52 amino acids, and serves as a key regulator of myocardial contractile kinetics (1-3). This regulation involves physical binding of PLB to the SERCA2a isoform of Ca 2ϩ -ATPase (4 -7), the Ca 2ϩ pump found in cardiac SR, which is responsible for actively transporting Ca 2ϩ into the SR lumen to maintain SR Ca 2ϩ stores. In particular, dephosphorylated PLB decreases the apparent affinity of the Ca 2ϩ pump for Ca 2ϩ by slowing the E2 to E1 transition of the catalytic cycle that couples ATP hydrolysis to Ca 2ϩ transport (8). Upon phosphorylation, this inhibitory effect of PLB on SERCA2a Ca 2ϩ affinity is reversed, resulting in increased Ca 2ϩ transport into the SR and in stronger heart contraction and relaxation, thus producing the so-called positive inotropic and lusitropic effects of ␤-adrenergic stimulation (1, 2). PLB contains two major domains, a regulatory, cytoplasmic domain I (residues 1-31), and an anchoring, transmembrane domain II (residues 32-52). At the cytoplasmic domain, several residues are important for PLB inhibitory function (9, 10). For example, Ser 16 and Thr 17 located ...

Abstract-Cardiac calsequestrin-null mice (Casq2 Ϫ/Ϫ ) display catecholaminergic ventricular tachycardia akin to humans with CASQ2 mutations. However, the specific contribution of Casq2 deficiency to the arrhythmia phenotype is difficult to assess because Casq2 Ϫ/Ϫ mice also show significant reductions in the sarcoplasmic reticulum (SR) proteins junctin and triadin-1 and increased SR volume. Furthermore, it remains unknown whether Casq2 regulates SR Ca 2ϩ release directly or indirectly by buffering SR luminal Ca 2ϩ . To address both questions, we examined heterozygous (Casq2 ϩ/Ϫ ) mice, which have a 25% reduction in Casq2 but no significant decrease in other SR proteins. Casq2