Junctin and calsequestrin overexpression in cardiac muscle: the role of junctin and the synthetic and delivery pathways for the two proteins

Ca(2+) storage and release in muscle cells are controlled by a complex of junctional sarcoplasmic reticulum (jSR) proteins, that includes the calcium-binding protein calsequestrin (CSQ), the Ca(2+)-release channel (ryanodine receptor or RyR) and two transmembrane proteins that bind to RyR: junctin (JNC) and triadin (Tr). The relationship between CSQ and JNC, and their contributions to the architecture of the jSR vesicle was studied in transgenic mice with combined overexpression of CSQ and JNC. We find that CSQ, on its own, has a diffuse disposition in the sarcoplasmic reticulum (SR) lumen. Overexpression of JNC results in a tighter packing of CSQ in proximity of the SR membrane, presumably due to the binding of CSQ to the membrane by JNC. Quantitative and qualitative analysis of structural changes in the overexpressing as well as in the normally differentiating myocardium illustrate the synthetic pathways and the events in the targeting and delivery of CSQ and JNC to the jSR of the differentiating cardiac myocyte. CSQ is delivered from the Golgi to the SR, where it buds out into precursors of the jSR vesicles. JNC reaches the jSR vesicles directly, but its arrival is delayed relative to CSQ.     

Sarcoplasmic reticulum Ca2+ release in neonatal rat cardiac myocytes

In the neonatal mammalian heart, the role of ryanodine receptor (= Ca²⁺ release channel)-mediated sarcoplasmic reticulum (SR) Ca²⁺ 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. Measurement of Ca²⁺ transients of infected cardiac myocytes revealed unchanged peak amplitudes under basal conditions but with overexpression of calsequestrin and triadin caffeine-releasable SR Ca²⁺ content was increased. Our results demonstrate that an increased expression of triadin or calsequestrin is associated with an increased SR Ca²⁺ storage but unchanged Ca²⁺ signaling in neonatal rat cardiac myocytes. This is consistent with an ancillary role of the sarcoplasmic reticulum in excitation–contraction coupling in the developing mammalian heart.     

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. We assessed arrhythmias and electrophysiological changes in vivo and in the beating heart in mice expressing junctin, triadin, or both proteins (TRDxJCN), and measured calcium transients in isolated ventricular cardiomyocytes. TRDxJCN mice were studied to compensate the down-regulation of junctin expression in triadin-expressing mice. Exercise or stress provoked repetitive VT in freely roaming TRDxJCN mice whenever heart rate increased above approximately 600 bpm (p<0.05 vs. the three other genotypes). TRDxJCN mice expressed total triadin 2.9-fold (p<0.05) and total junctin not different to wildtype (p=ns). Left ventricular systolic function was not different between lineages. beta-adrenoreceptor stimulation (orciprenaline 1.7 microM) provoked early-coupled ventricular ectopy and repetitive VT in isolated, Langendorff-perfused TRDxJCN hearts (p<0.05). Under conditions associated with VT (high pacing rate, catecholamine stimulation), action potential duration was shorter in TRDxJCN with VT than in the other genotypes and shorter than in TRDxJCN hearts without VT (p<0.05). Ca(2+) transient duration was prolonged in Indo1-loaded TRDxJCN cardiomyocytes under VT-provoking conditions. Action potential prolongation by mexiletine (2 microM or 4 microM) or clarithromycine (150 microM) suppressed VT. Expression of triadin provokes stress- and tachycardia-related ventricular arrhythmias in mice. An imbalance between prolonged intracellular calcium release and shortening of the ventricular action potential may contribute to genesis of arrhythmias in this model.     

Overexpression of junctate induces cardiac hypertrophy and arrhythmia via altered calcium handling

Junctate-1 is a newly identified integral endoplasmic/sarcoplasmic reticulum Ca²⁺ binding protein. However, its functional role in the heart is unknown. In the present study, the consequences of constitutively overexpressed junctate in cardiomyocytes were investigated using transgenic (TG) mice overexpressing junctate-1. TG mice (8 weeks old) showed cardiac remodeling such as marked bi-atrial enlargement with intra-atrial thrombus and biventricular hypertrophy. The TG mice also showed bradycardia with atrial fibrillation, reduced amplitude and elongated decay time of Ca²⁺ transients, increased L-type Ca²⁺ current and prolonged action potential durations. Time-course study (2-8 weeks) showed an initially reduced SR function due to down-regulation of SERCA2 and calsequestrin followed by sarcolemmal protein expression and cardiac hypertrophy at later age. These sequential changes could well be correlated with the physiological changes. Adrenergic agonist treatment and subsequent biochemical study showed that junctate-1 TG mice (8 weeks old) were under local PKA signaling that could cause increased L-type Ca²⁺ current and reduced SR function. Junctate-1 in the heart is closely linked to the homeostasis of E-C coupling proteins and a sustained increase of junctate-1 expression leads to a severe cardiac remodeling and arrhythmias.