Ca2+ release from sarcoplasmic reticulem in cardiac cells during excitation-contraction coupling is inatiated by Ca2+ influx through L-type Ca2+ channels. We recently demonstrated the feasibility to visualize Ca2+ influxes of single L-type Ca2+ channel (LCC) openings- """"""""Ca2+ sparklets""""""""- using confocal linescan imaging combined with patch clamp techniques. Here we directly visualize the process that Ca2+ sparklets trigger Ca2+ sparks in rat ventricular myocytes. To preserve the delicate molecular architecture of E-C coupling, loose-patch clamp is established by gently pressing the patch pipette against cell surface without suction. In the presence of 10 microM FPL64176 and 20 mM [Ca2+]o, depolarization pulses (-40 to +50 mV) evoke local Ca2+ transients that fall into two distinct categories. The low-amplitude events with irregular duration were resistant to ryanodine (10 mM), but were abolished by 2 mM nifedipine, and thus represent Ca2+ sparklets. The high-amplitude events were identified as Ca2+ sparks by virtue of morphology and sensitivity to ryanodine. The blockade of Ca2+ sparklets by substitution of Ca2+ with Ba2+ in the pipette abolished the Ca2+ spark activity, indicating that the sparks are triggered by sparklets. Our data provide direct evidence that Ca2+ flux from single L-type channel is capable of triggering a Ca2+ spark. The simultaneous recording of sparklets and sparks make it possible to probe into kinetics of cardiac excitation-contraction coupling at the single-channel level. During 400-ms depolarising pulses, 71% of the first local Ca2+ events occurred as triggered sparks, suggesting that a Ca2+ sparklet does not always elicit a spark. When a sparklet did trigger a spark, the latency varied widely from case to case. Interestingly, the coupling fidelity was significantly reduced to 30% after the first triggered spark, reflecting a local refractoriness of SR Ca2+ release. These evidence suggest that the LCC-RyR coupling is probabilistic rather than deterministic by nature, and that the coupling efficiency varies in a use-dependent manner. Ca2+ sparklets of known iCa also offer a natural standard to gauge the SR Ca2+ efflux underlying the spark (jspark). Upon repolerization to -70 mV, tail current-evoked sparklets displayed a rising time constant of 9.3 ms and a steady-state amplitude of 57 nM. By comparison, an averaged Ca2+ spark exhibited a peak amplitude that was 3.1-fold greater than the sparklet at the same rising time. Assuming a linear relation between Ca2+ flux and local Ca2+ transient, we estimated that the average jspark mounts to 2.1 pA, suggesting that 4 ~ 6 RyRs are tipically involved in an evoked Ca2+ spark. Altogether, our work provide direct evidence that Ca2+ sparklets due to single LCCs constitute fundamental trigger events of SR Ca2+ release, which ignite elemental Ca2+ sparks originated from 4 ~ 6 RyRs. We found that cardiac E-C coupling at the molecular level is inherently stochastic. The optical analysis of single-channel physiology and intermolecular crosstalk affords a powerful means for elucidation of Ca2+ signalling mechanisms at the molecular level.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Intramural Research (Z01)
Project #
Application #
Study Section
National Institute on Aging Initial Review Group (NIA)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
United States
Zip Code
Zhu, Wei-Zhong; Chakir, Khalid; Zhang, Shengjun et al. (2005) Heterodimerization of beta1- and beta2-adrenergic receptor subtypes optimizes beta-adrenergic modulation of cardiac contractility. Circ Res 97:244-51
Song, Long-Sheng; Pi, Yeqing; Kim, Song-Jung et al. (2005) Paradoxical cellular Ca2+ signaling in severe but compensated canine left ventricular hypertrophy. Circ Res 97:457-64
Xiao, Rui-Ping; Zhu, Weizhong; Zheng, Ming et al. (2004) Subtype-specific beta-adrenoceptor signaling pathways in the heart and their potential clinical implications. Trends Pharmacol Sci 25:358-65
Ding, Jian-Hua; Xu, Xiangdong; Yang, Dongmei et al. (2004) Dilated cardiomyopathy caused by tissue-specific ablation of SC35 in the heart. EMBO J 23:885-96
Wang, Wang; Zhu, Weizhong; Wang, Shiqiang et al. (2004) Sustained beta1-adrenergic stimulation modulates cardiac contractility by Ca2+/calmodulin kinase signaling pathway. Circ Res 95:798-806
Wang, Shi-Qiang; Wei, Chaoliang; Zhao, Guiling et al. (2004) Imaging microdomain Ca2+ in muscle cells. Circ Res 94:1011-22
Yang, Xiaoling; Wang, Wengong; Fan, Jinshui et al. (2004) Prostaglandin A2-mediated stabilization of p21 mRNA through an ERK-dependent pathway requiring the RNA-binding protein HuR. J Biol Chem 279:49298-306
Stern, Michael D; Cheng, Heping (2004) Putting out the fire: what terminates calcium-induced calcium release in cardiac muscle? Cell Calcium 35:591-601
Wang, Shi Qiang; Stern, Michael D; Rios, Eduardo et al. (2004) The quantal nature of Ca2+ sparks and in situ operation of the ryanodine receptor array in cardiac cells. Proc Natl Acad Sci U S A 101:3979-84
Jiang, Dawei; Xiao, Bailong; Yang, Dongmei et al. (2004) RyR2 mutations linked to ventricular tachycardia and sudden death reduce the threshold for store-overload-induced Ca2+ release (SOICR). Proc Natl Acad Sci U S A 101:13062-7

Showing the most recent 10 out of 15 publications