ABSTRACT
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. Measurement of Ca(2+) transients of infected cardiac myocytes revealed unchanged peak amplitudes under basal conditions but with overexpression of calsequestrin and triadin caffeine-releasable SR Ca(2+) content was increased. Our results demonstrate that an increased expression of triadin or calsequestrin is associated with an increased SR Ca(2+) storage but unchanged Ca(2+) 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.
Subject(s)
Calcium-Binding Proteins/metabolism , Calcium/metabolism , Excitation Contraction Coupling/physiology , Ion Transport/physiology , Myocytes, Cardiac/metabolism , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/metabolism , Adenoviridae , Animals , Animals, Newborn , Caffeine/pharmacology , Calcium Signaling/physiology , Calcium-Binding Proteins/genetics , Calsequestrin/genetics , Calsequestrin/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Excitation Contraction Coupling/drug effects , Gene Expression Regulation , Genetic Vectors , Heart/drug effects , Heart/physiology , Ion Transport/drug effects , Muscle Proteins/genetics , Muscle Proteins/metabolism , Myocardial Contraction/drug effects , Myocardial Contraction/physiology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Transduction, GeneticABSTRACT
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. Junctin was below our level of detection in SR membranes from failing human hearts, and triadin was downregulated by 22%. To better understand the role of junctin in the regulation of Ca(2+) homeostasis and contraction of cardiac myocytes, we used an adenoviral approach to overexpress junctin in isolated rat cardiac myocytes. A recombinant adenovirus encoding the green fluorescent protein served as a control. Infection of myocytes with the junctin-expressing virus resulted in an increased RNA and protein expression of junctin. Ca(2+) transients showed a decreased maximum Ca(2+) amplitude, and contractility of myocytes was depressed. Our results demonstrate that an increased expression of junctin is associated with an impaired Ca(2+) homeostasis. Downregulation of junctin in human heart failure may thus be a compensatory mechanism.
