Critical Sorting Steps and Pathways in the Trafficking of Cardiac Sarcoplasmic Reticulum Proteins

Cardiac hypertrophy can lead to heart failure, with few treatment options. Hypertrophic growth inevitably
requires mechanisms of protein distribution, as studies show the involvement of both increased mRNA
translation and mRNA trafficking along microtubules. Yet, how the distribution of membrane proteins occurs and
responds to hypertrophic growth is unknown. While sarcoplasmic reticulum (SR) Ca handling is key to
hypertrophy, we do not know how trafficking of SR proteins occurs using cardiomyocyte specific mechanisms,
nor its regulation and response to hypertrophy, which precludes discovery of key targets for therapeutics. To
probe this mechanism and identify sites of regulation, we have focused on a common portion of the biosynthetic
membrane pathway where the rough endoplasmic reticulum (ER) organizes and sorts proteins for transit into
SR tubules. Using a species-specific expression and immunological analysis to study the early steps of SR
protein trafficking in adult cardiomyocytes, we show that newly synthesized proteins first accumulate into
organized perinuclear ER puncta aligned with z-lines, and then transit to transverse SR z-tubules that aligned
with T-tubules. Such morphology is observed for all newly made proteins examined, except for phospholamban,
suggesting commonality of these protein sorting sites in ER. Afterward, motors can power transport of proteins
both radially and axially via microtubules to their steady state distributions. These data lead to our hypotheses
that newly synthesized SR proteins are sorted in a specific set of perinuclear ER subdomains, which are
organized by the alignment with z-lines and in proximity to nuclear pores and translocons, contain critical ER
trafficking proteins, and allow newly made SR proteins to develop their initial functional interactions for transit to
SR. Such cellular sorting and trafficking steps adapt to the physiological demands, but maladapt to hypertrophic
heart failure, which are likely amenable to develop new therapeutics regulating membrane protein trafficking. To
dissect biochemical features of these sorting sites and trafficking steps, we have developed key experimental
systems to determine progressive protein distribution 12-48 h after translation of three small single
transmembrane domain proteins that are destined to different subcellular locations. Included is phospholamban
(in the nuclear envelope and in longitudinal SR for Ca uptake), junctin (in junctional SR for Ca release), and
phospholemman (in sarcolemma and intercalated discs to regulate the Na, K-pump). Aim 1 will determine: if a
single perinuclear ER subdomain can effectively accumulate and co-localize newly made SR proteins (junctin
and phospholamban), but separate phospholemman, for their diverse and distinct steady state distributions; if
sites are aligned with nuclear pores, translocons; and if known ER trafficking proteins, newly identified in our
SERCA-activated SR proteome, will modulate these trafficking steps. Aim 2 will determine whether a
hypertrophic response modulates the accumulations of protein into any of the ER sorting sites, their common
biosynthesis pathways, or the novel sets of cardiac ER trafficking proteins, in animal models of hypertrophy.

Grant Number: 5R01HL169877-04
NIH Institute/Center: NIH
Principal Investigator: Zhenhui Chen