A growing body of evidence, including studies using genetically engineered mice, has shown that Ca²⁺ cycling and Ca²⁺-dependent signaling pathways play a pivotal role in cardiac hypertrophy and heart failure. We previously reported that transgenic (TG) mice overexpressing calsequestrin (JBC 1999;274:22251), juntin (FASEB J 2002;16:1310) or junctate-1 (JACC 2003;41:313A) demonstrated cardiac hypertrophy and failure, abnormal calcium handling and arrhythmias. To elucidate the interplay of Ca²⁺ handling proteins and pathogenetic mechanism of electrical abnormalities (bradycardia, atrial fibrillation, ventricular premature beats, sinus pause) in the junctate-1 TG heart, we evaluated expression levels of excitation-contraction coupling proteins and measured action potential, Ca²⁺ currents and Ca²⁺ release from SR in the isolated single cardiomyocytes. Overexpression of junctate-1 led to down-regulation of SR Ca²⁺-ATPase 2 calsequestrin, calreticulin and ryanodine receptor but to up-regulation of Na⁺-Ca²⁺ exchanger and plasma membrane Ca²⁺-ATPase. The expression levels of triadin, junctin, phospholamban, and dihydropyridine receptor did not change significantly. The density of ryanodine receptor, as determined by Bmax of ryanodine to the receptor, were significantly lower in TG hearts than in wild-type heart (0.53짹0.04 vs 0.81짹0.02 pmol/mg protein, p<0.05), whereas the Kd of ryanodine binding was not significantly altered (wild-type, 1.14짹0.04; TG, 1.07짹0.09 nM). The SR Ca²⁺ content decreased whereas the L-type Ca²⁺ current density increased in the isolated ventricular cardiomyocytes from junctate-1 transgenic heart. The resting membrane potential (65짹5 mV) was not changed (wild-type, 67짹5 mV), but the action potential duration at 90% repolarization increased significantly to 174짹12 ms (wild-type, 50짹9 ms, p<0.05) by the overexpression of junctate-1 to the ventricular muscle. The present work has provided an important example of pathogenesis leading to cardiac hypertrophy and arrhythmia, which was due to impaired Ca²⁺ handling induced by overexpression of junctate-1 in heart.