Open Access Dissertation
Date of Award
Junctional SR is an important and unique ER subdomain in the adult myocyte that releases Ca2+ through the actions of an exclusive set of resident proteins. Cardiac calsequestrin (CSQ2) undergoes two co-translational modifications: N-linked glycosylation on 316Asn, and phosphorylation by protein kinase CK2 on a cluster of 3 serines in its tail. In the heart, CSQ2 molecules undergo extensive mannose trimming by ER mannosidase(s), a posttranslational process that often regulates protein breakdown. To investigate CSQ2 protein processing in cardiomyopathy models, studies were performed to test whether CSQ2 glycan structures would be altered in heart tissue from mongrel dogs induced into heart failure (HF) by two different experimental treatments, rapid ventricular pacing or repeated coronary microembolizations. CSQ2 mRNA and protein levels were determined, and its processing was analyzed by electrospray mass spectrometry. Despite the different cardiomyopathies producing the failure, roughly one-third of CSQ2 molecules from each of the failed tissues contained glycan structures indicating a buildup of newly made protein with highly reduced glycan trimming. Analyses of tissue samples showed decreases in CSQ2 protein levels per unit levels of mRNA for tachypaced heart tissue, also indicative of altered turnover.
To place these data in context of junctional SR cell biology, we carried out a series of studies on primary rat cardiomyocytes to understand the basic features of junctional SR protein trafficking. We expressed canine forms of junctin (JCT), triadin (TRD) and CSQ2 in adult rat cardiomyocytes, each a major protein component of the Ca2+ release complex. Two additional reagents employed were CSQ2-DsRed, which fails to leave sites of early synthesis due to immediate polymerization, and so was able to bind TRD and JCT in rough ER; and a deletion mutant of TRD (delTRD) which was not so affected, and allowed us to directly probe the differences between ER and SR by double labeling. A complex picture emerged, in which all junctional SR proteins are synthesized in the nuclear envelope but travel across MT linked smooth ER to continuously deposit junctional SR proteins. The data suggest that ER proteins traffic radially along Z-lines, and axially between nuclei, maintaining junctional SR protein levels through sorting and selective retention as ER traverses Z-lines. ER protein traffic requires intact MTs, as nocodazole treatment led to loss of anterograde JCT and TRD traffic away from the nuclear envelope.
We conclude that altered processing of CSQ2 may be an adaptive response to the myocardium under stresses that are capable of inducing heart failure. We provide a model of ER and SR protein transport across the cardiomyocytes, and a new understanding of pathways by which cellular Ca2+ handling protein complexes can be maintained and modified.
Sleiman, Naama, "Junctional Sarcoplasmic Reticulum Protein Processing And Trafficking In Cardiac Tissue And Primary Cultured Cardiomyocytes" (2014). Wayne State University Dissertations. 1056.