Transitions of protein traffic from cardiac ER to junctional SR.

The junctional sarcoplasmic reticulum (jSR) is an important and unique ER subdomain in the adult myocyte that concentrates resident proteins to regulate Ca(2+) release. To investigate cellular mechanisms for sorting and trafficking proteins to jSR, we overexpressed canine forms of junctin (JCT) or triadin (TRD) in adult rat cardiomyocytes. Protein accumulation over time was visualized by confocal fluorescence microscopy using species-specific antibodies.

Acute reversal of phospholamban inhibition facilitates the rhythmic whole-cell propagating calcium waves in isolated ventricular myocytes.

Phospholamban (PLB) inhibits the activity of cardiac sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a). Phosphorylation of PLB during sympathetic activation reverses SERCA2a inhibition, increasing SR Ca(2+) uptake. However, sympathetic activation also modulates multiple other intracellular targets in ventricular myocytes (VMs), making it impossible to determine the specific effects of the reversal of PLB inhibition on the spontaneous SR Ca(2+) release.

Sarcoplasmic reticulum Ca2+ cycling protein phosphorylation in a physiologic Ca2+ milieu unleashes a high-power, rhythmic Ca2+ clock in ventricular myocytes: relevance to arrhythmias and bio-pacemaker design.

Basal phosphorylation of sarcoplasmic reticulum (SR) Ca(2+) proteins is high in sinoatrial nodal cells (SANC), which generate partially synchronized, spontaneous, rhythmic, diastolic local Ca(2+) releases (LCRs), but low in ventricular myocytes (VM), which exhibit rare diastolic, stochastic SR-generated Ca(2+) sparks. We tested the hypothesis that in a physiologic Ca(2+) milieu, and independent of increased Ca(2+) influx, an increase in basal phosphorylation of SR Ca(2+) cycling proteins will convert stochastic Ca(2+) sparks into periodic, high-power Ca(2+) signals of the type that drives SANC normal automaticity. We measured phosphorylation of SR-associated proteins, phospholamban (PLB) and ryanodine receptors (RyR), and spontaneous local Ca(2+) release characteristics (LCR) in permeabilized single, rabbit VM in physiologic [Ca(2+)], prior to and during inhibition of protein phosphatase (PP) and phosphodiesterase (PDE), or addition of exogenous cAMP, or in the presence of an antibody (2D12), that specifically inhibits binding of the PLB to SERCA-2.

The structural basis for phospholamban inhibition of the calcium pump in sarcoplasmic reticulum.

P-type ATPases are a large family of enzymes that actively transport ions across biological membranes by interconverting between high (E1) and low (E2) ion-affinity states; these transmembrane transporters carry out critical processes in nearly all forms of life. In striated muscle, the archetype P-type ATPase, SERCA (sarco(endo)plasmic reticulum Ca(2+)-ATPase), pumps contractile-dependent Ca(2+) ions into the lumen of sarcoplasmic reticulum, which initiates myocyte relaxation and refills the sarcoplasmic reticulum in preparation for the next contraction. In cardiac muscle, SERCA is regulated by phospholamban (PLB), a small inhibitory phosphoprotein that decreases the Ca(2+) affinity of SERCA and attenuates contractile strength.

Altered calsequestrin glycan processing is common to diverse models of canine heart failure.

Calsequestrin-2 (CSQ2) is a resident glycoprotein of junctional sarcoplasmic reticulum that functions in the regulation of SR Ca(2+) release. CSQ2 is biosynthesized in rough ER around cardiomyocyte nuclei and then traffics transversely across SR subcompartments. During biosynthesis, CSQ2 undergoes N-linked glycosylation and phosphorylation by protein kinase CK2.

Characterizing phospholamban to sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) protein binding interactions in human cardiac sarcoplasmic reticulum vesicles using chemical cross-linking.

Chemical cross-linking was used to study protein binding interactions between native phospholamban (PLB) and SERCA2a in sarcoplasmic reticulum (SR) vesicles prepared from normal and failed human hearts. Lys(27) of PLB was cross-linked to the Ca(2+) pump at the cytoplasmic extension of M4 (at or near Lys(328)) with the homobifunctional cross-linker, disuccinimidyl glutarate (7.7 Å).

Sarcoplasmic reticulum Ca2+ release in neonatal rat cardiac myocytes.

In the neonatal mammalian heart, the role of ryanodine receptor (=Ca(2+) release channel)-mediated sarcoplasmic reticulum (SR) Ca(2+) release for excitation-contraction coupling is still a matter of debate. Using an adenoviral system, we overexpressed separately the junctional SR proteins triadin, junctin, and calsequestrin, which are probably involved in regulation of ryanodine receptor function. Infection of neonatal rat cardiac myocytes with triadin, junctin, or calsequestrin viruses, controlled by green fluorescent protein expression, resulted in an increased protein level of the corresponding transgenes.

Losartan reduces mortality in a genetic model of heart failure.

Altered Ca(2+) homoeostasis accompanies heart failure. As a model of heart failure, transgenic mice (TG) with selective overexpression of calsequestrin (CSQ) in the heart were used. CSQ is the main Ca(2+) binding protein in the lumen of the junctional sarcoplasmic reticulum.

Superinhibitory phospholamban mutants compete with Ca2+ for binding to SERCA2a by stabilizing a unique nucleotide-dependent conformational state.

Three cross-linkable phospholamban (PLB) mutants of increasing inhibitory strength (N30C-PLB < N27A,N30C,L37A-PLB (PLB3) < N27A,N30C,L37A,V49G-PLB (PLB4)) were used to determine whether PLB decreases the Ca(2+) affinity of SERCA2a by competing for Ca(2+) binding. The functional effects of N30C-PLB, PLB3, and PLB4 on Ca(2+)-ATPase activity and E1 approximately P formation were correlated with their binding interactions with SERCA2a measured by chemical cross-linking. Successively higher Ca(2+) concentrations were required to both activate the enzyme co-expressed with N30C-PLB, PLB3, and PLB4 and to dissociate N30C-PLB, PLB3, and PLB4 from SERCA2a, suggesting competition between PLB and Ca(2+) for binding to SERCA2a.

Changes in metabolic profile and population of skeletal muscle fibers of mice overexpressing calsequestrin: influence of losartan.

In heart failure, exertional fatigue of skeletal muscles can occur. A transgenic mouse overexpressing calsequestrin can be regarded as an animal model of heart failure. The aims of the present study were to investigate, whether at the time of cardiac failure the composition of fiber types of skeletal muscles was altered, what kind of alterations in glycolytic and oxidative enzyme activities occurred in different muscle fiber types and whether these were affected by the administration of the angiotensin II receptor blocker, losartan.

Ca2+ binding to site I of the cardiac Ca2+ pump is sufficient to dissociate phospholamban.

Phospholamban (PLB) inhibits the activity of SERCA2a, the Ca(2+)-ATPase in cardiac sarcoplasmic reticulum, by decreasing the apparent affinity of the enzyme for Ca(2+). Recent cross-linking studies have suggested that PLB binding and Ca(2+) binding to SERCA2a are mutually exclusive. PLB binds to the E2 conformation of the Ca(2+)-ATPase, preventing formation of E1, the conformation that binds two Ca(2+) (at sites I and II) with high affinity and is required for ATP hydrolysis.

Ablation of triadin causes loss of cardiac Ca2+ release units, impaired excitation-contraction coupling, and cardiac 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.

Intracellular calcium dynamics and acceleration of sinus rhythm by beta-adrenergic stimulation.

Background: Recent evidence indicates that membrane voltage and Ca2+ clocks jointly regulate sinoatrial node (SAN) automaticity. Here we test the hypothesis that sinus rate acceleration by beta-adrenergic stimulation involves synergistic interactions between these clock mechanisms.

Methods And Results: We simultaneously mapped intracellular calcium (Ca(i)) and membrane potential in 25 isolated canine right atrium, using previously described criteria of the timing of late diastolic Ca(i) elevation (LDCAE) relative to the action potential upstroke to detect the Ca2+ clock.

Triadins modulate intracellular Ca(2+) homeostasis but are not essential for excitation-contraction coupling in skeletal muscle.

To unmask the role of triadin in skeletal muscle we engineered pan-triadin-null mice by removing the first exon of the triadin gene. This resulted in a total lack of triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype.

Modest reductions of cardiac calsequestrin increase sarcoplasmic reticulum Ca2+ leak independent of luminal Ca2+ and trigger ventricular arrhythmias in mice.

Cardiac calsequestrin-null mice (Casq2-/-) 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-/- 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 Ca2+ release directly or indirectly by buffering SR luminal Ca2+.

Mechanism of reversal of phospholamban inhibition of the cardiac Ca2+-ATPase by protein kinase A and by anti-phospholamban monoclonal antibody 2D12.

Our model of phospholamban (PLB) regulation of the cardiac Ca(2+)-ATPase in sarcoplasmic reticulum (SERCA2a) states that PLB binds to the Ca(2+)-free, E2 conformation of SERCA2a and blocks it from transitioning from E2 to E1, the Ca(2+)-bound state. PLB and Ca(2+) binding to SERCA2a are mutually exclusive, and PLB inhibition of SERCA2a is manifested as a decreased apparent affinity of SERCA2a for Ca(2+). Here we extend this model to explain the reversal of SERCA2a inhibition that occurs after phosphorylation of PLB at Ser(16) by protein kinase A (PKA) and after binding of the anti-PLB monoclonal antibody 2D12, which recognizes residues 7-13 of PLB.

Stress and high heart rate provoke ventricular tachycardia in mice expressing triadin.

Reduced function of the cardiac ryanodine receptor or calsequestrin causes catecholaminergic ventricular tachycardia (VT). These proteins regulate sarcoplasmic Ca(2+) release in close conjunction with two accessory proteins, triadin and junctin. Based on data from cardiomyocytes, we hypothesized that enhanced triadin expression could cause VT.

On the role of junctin in cardiac Ca2+ handling, contractility, and heart failure.

Junctin is a transmembrane protein located at the cardiac junctional sarcoplasmic reticulum (SR) and forms a quaternary complex with the Ca(2+) release channel, triadin and calsequestrin. Impaired protein interactions within this complex may alter the Ca(2+) sensitivity of the Ca(2+) release channel and may lead to cardiac dysfunction, including hypertrophy, depressed contractility, and abnormal Ca(2+) transients. To study the expression of junctin and, for comparison, triadin, in heart failure, we measured the levels of these proteins in SR from normal and failing human hearts.

Sarcoplasmic reticulum calcium overloading in junctin deficiency enhances cardiac contractility but increases ventricular automaticity.

Background: Abnormal sarcoplasmic reticulum calcium (Ca) cycling is increasingly recognized as an important mechanism for increased ventricular automaticity that leads to lethal ventricular arrhythmias. Previous studies have linked lethal familial arrhythmogenic disorders to mutations in the ryanodine receptor and calsequestrin genes, which interact with junctin and triadin to form a macromolecular Ca-signaling complex. The essential physiological effects of junctin and its potential regulatory roles in sarcoplasmic reticulum Ca cycling and Ca-dependent cardiac functions, such as myocyte contractility and automaticity, are unknown.

Casq2 deletion causes sarcoplasmic reticulum volume increase, premature Ca2+ release, and catecholaminergic polymorphic ventricular tachycardia.

Cardiac calsequestrin (Casq2) is thought to be the key sarcoplasmic reticulum (SR) Ca2+ storage protein essential for SR Ca2+ 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.

Cross-linking of C-terminal residues of phospholamban to the Ca2+ pump of cardiac sarcoplasmic reticulum to probe spatial and functional interactions within the transmembrane domain.

Interactions between the transmembrane domains of phospholamban (PLB) and the cardiac Ca2+ 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 Cu2+-phenanthroline, dibromobimane, and bismaleimidohexane. V49C-, M50C-, and L52C-PLB all cross-linked strongly to V89C-SERCA2a, coupling to 70-100% of SERCA2a molecules.

Overexpression of junctin causes adaptive changes in cardiac myocyte Ca(2+) signaling.

In cardiac muscle, junctin forms a quaternary protein complex with the ryanodine receptor (RyR), calsequestrin, and triadin 1 at the luminal face of the junctional sarcoplasmic reticulum (jSR). By binding directly the RyR and calsequestrin, junctin may mediate the Ca(2+)-dependent regulatory interactions between both proteins. To gain more insight into the underlying mechanisms of impaired contractile relaxation in transgenic mice with cardiac-specific overexpression of junctin (TG), we studied cellular Ca(2+) handling in these mice.

Role of leucine 31 of phospholamban in structural and functional interactions with the Ca2+ pump of cardiac sarcoplasmic reticulum.

The ability of two loss-of-function mutants, L31A and L31C, of phospholamban (PLB) to bind to and inhibit the Ca(2+) pump of cardiac sarcoplasmic reticulum (SERCA2a) was investigated using a molecular cross-linking approach. Leu(31) of PLB, located at the cytoplasmic membrane boundary, is a critical amino acid shown previously to be essential for Ca(2+)-ATPase inhibition. We observed that L31A or L31C mutations of PLB prevented the inhibition of Ca(2+)-ATPase activity and disabled the cross-linking of N27C and N30C of PLB to Lys(328) and Cys(318) of SERCA2a.

Hypertrophy, increased ejection fraction, and reduced Na-K-ATPase activity in phospholemman-deficient mice.

Phospholemman (FXYD1), a 72-amino acid transmembrane protein abundantly expressed in the heart and skeletal muscle, is a major substrate for phosphorylation in the cardiomyocyte sarcolemma. Biochemical, cellular, and electrophysiological studies have suggested a number of possible roles for this protein, including ion channel modulator, taurine-release channel, Na(+)/Ca(2+) exchanger modulator, and Na-K-ATPase-associated subunit. We have generated a phospholemman-deficient mouse.