Article

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

Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University, Nashville, TN 37232, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 05/2009; 106(18):7636-41. DOI: 10.1073/pnas.0902919106
Source: PubMed

ABSTRACT 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 (I(Ca)). Uninhibited Ca(2+) influx via I(Ca) likely contributes to Ca(2+) overload and results in spontaneous SR Ca(2+) releases upon beta-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.

0 Bookmarks
 · 
196 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The histidine-rich Ca(2+)-binding protein (HRC) is located in the lumen of the sarcoplasmic reticulum (SR) and exhibits high-capacity Ca(2+)-binding properties. Overexpression of HRC in the heart resulted in impaired SR Ca(2+) uptake and depressed relaxation through its interaction with SERCA2a. However, the functional significance of HRC in overall regulation of calcium cycling and contractility is not currently well defined. To further elucidate the role of HRC in vivo under physiological and pathophysiological conditions, we generated and characterized HRC-knockout (KO) mice. The KO mice were morphologically and histologically normal compared to wild-type (WT) mice. At the cellular level, ablation of HRC resulted in significantly enhanced contractility, Ca(2+) transients, and maximal SR Ca(2+) uptake rates in the heart. However, after-contractions were developed in 50 % of HRC-KO cardiomyocytes, compared to 11 % in WT mice under stress conditions of high-frequency stimulation (5 Hz) and isoproterenol application. A parallel examination of the electrical activity revealed significant increases in the occurrence of Ca(2+) spontaneous SR Ca(2+) release and delayed afterdepolarizations with ISO in HRC-KO, compared to WT cells. The frequency of Ca(2+) sparks was also significantly higher in HRC-KO cells with ISO, consistent with the elevated SR Ca(2+) load in the KO cells. Furthermore, HRC-KO cardiomyocytes showed significantly deteriorated cell contractility and Ca(2+)-cycling caused possibly by depressed SERCA2a expression after transverse-aortic constriction (TAC). Also HRC-null mice exhibited severe cardiac hypertrophy, fibrosis, pulmonary edema and decreased survival after TAC. Our results indicate that ablation of HRC is associated with poorly regulated SR Ca(2+)-cycling, and severe pathology under pressure-overload stress, suggesting an essential role of HRC in maintaining the integrity of cardiac function.
    Archiv für Kreislaufforschung 05/2013; 108(3):344. · 7.35 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cardiac calsequestrin (CASQ2) contributes to intracellular Ca(2+) homeostasis by virtue of its low-affinity/high-capacity Ca(2+) binding properties, maintains sarcoplasmic reticulum (SR) architecture and regulates excitation-contraction coupling, especially or exclusively upon β-adrenergic stimulation. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease associated with cardiac arrest in children or young adults. Recessive CPVT variants are due to mutations in the CASQ2 gene. Molecular and ultra-structural properties were studied in hearts of CASQ2(R33Q/R33Q) and of CASQ2(-/-) mice from post-natal day 2 to week 8. The drastic reduction of CASQ2-R33Q is an early developmental event and is accompanied by down-regulation of triadin and junctin, and morphological changes of jSR and of SR-transverse-tubule junctions. Although endoplasmic reticulum stress is activated, no signs of either apoptosis or autophagy are detected. The other model of recessive CPVT, the CASQ2(-/-) mouse, does not display the same adaptive pattern. Expression of CASQ2-R33Q influences molecular and ultra-structural heart development; post-natal, adaptive changes appear capable of ensuring until adulthood a new pathophysiological equilibrium.
    Experimental Cell Research 12/2013; · 3.56 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Junctophilin-2 (JP2) is believed to hold the t-tubular and junctional sarcoplasmic reticulum (jSR) membranes in a precise geometry that facilitates excitation-contraction coupling in cardiomyocytes. We have expressed and purified human JP2 and shown using electron microscopy that the protein forms elongated structures ~ 15 nm long and 2 nm wide. Employing lipid binding assays and quartz crystal microbalance with dissipation we have determined that JP2 is selective for phosphatidylserine (PS), KD ~ 0.5 µM, with the N-terminal domain mediating this interaction. JP2 also binds phosphatidylinositol (3,4,5) triphosphate (PIP3), at a different site than PS, resulting in the protein adopting a more flexible conformation; this interaction is modulated by both Ca2+ and Mg2+ ions. We show that the S101R mutation identified in patients with hypertrophic cardiomyopathy leads to modification of the protein secondary structure, forming a more flexible molecule with an increased affinity for PS, but does not undergo a structural transition in response to binding PIP3. In conclusion this study provides new insights into the structural and lipid binding properties of JP2 and how the S101R mutation may impact upon the stability of the dyad organization with the potential to alter JP2-protein interactions regulating Ca2+ cycling.
    Biochemical Journal 09/2013; · 4.65 Impact Factor

Full-text

View
1 Download
Available from