The number and activity of Cav1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca2+-induced Ca2+ release and myocardial contraction. β-adrenergic receptor (βAR) activation stimulates sarcolemmal insertion of CaV1.2 channels. This supplements the pre-existing sarcolemmal CaV1.2 population, forming large ‘super-clusters’ wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca2+ influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early- and recycling endosome-localized, pre-synthesized CaV1.2 channels. βAR-activation decreased CaV1.2/endosome colocalization in ventricular myocytes, as it triggered ‘emptying’ of endosomal CaV1.2 cargo into the sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that CaV1.2 are often inserted into the sarcolemma as pre-formed, multi-channel clusters. Likewise, entire clusters were removed from the sarcolemma during endocytosis, while in other cases, a more incremental process suggested removal of individual channels. The amplitude of the stimulated insertion response was doubled by co-expression of constitutively-active Rab4a, halved by co-expression of dominant-negative Rab11a, and abolished by co-expression of dominant-negative mutant Rab4a. In ventricular myocytes, βAR-stimulated recycling of CaV1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented βAR-activated Ca2+ current augmentation. Moreover, βAR-regulation of CaV1.2 was abolished when recycling was halted by co-application of nocodazole and latrunculin-A. These findings reveal that βAR-stimulation triggers an on-demand boost in sarcolemmal CaV1.2 abundance via targeted, Rab4a and Rab11a-dependent insertion of CaV1.2 channels is essential for βAR-regulation of cardiac CaV1.2.Significance StatementThe L-type voltage-gated Ca2+ channel CaV1.2 is essential for excitation-contraction coupling in the heart. During the fight-or-flight response, CaV1.2 channel activity is augmented as a result of PKA-mediated phosphorylation, downstream of β-adrenergic receptor (βAR) activation. We discovered that enhanced sarcolemmal abundance of CaV1.2 channels, driven by stimulated insertion/recycling of specific CaV1.2 containing endosomes, is essential for βAR-mediated regulation of these channels in the heart. These data reveal a new conceptual framework of this critical and robust pathway for on-demand tuning of cardiac EC-coupling during fight-or-flight.

Mobisaic is a World Wide Web information system designed to serve users in a mobile wireless computing environment. Mobisaic extends the Web by allowing documents to both refer and react to potentially changing contextual information, such as current location in the wireless network. Mobisaic relies on clientside processing of HTML documents that support two new concepts: Dynamic Uniform Resource Locators URLs and Active Documents. A dynamic URL is one whose results depend upon the state of the user's mobile context at the time it is resolved. An active document is one that automatically updates its contents in response to changes in a user's mobile context. This paper describes the design of Mobisaic, the mechanism it uses for representing a user's mobile context, and the extensions made to the syntax and function of Uniform Resource L o c ators and HyperText Markup Language documents to support mobility.

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