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Visualizing Subcellular Localization of a Protein in the Heart Using Quantum Dots-Mediated Immuno-Labeling Followed by Transmission Electron Microscopy

The subcellular localization is critical to delineating proper function and determining the molecular mechanisms of a particular protein. Several ...

Louisiana State University Health Sciences Center-Shreveport

Methamphetamine-associated cardiomyopathy is the leading cause of death linked with illicit drug use. Here we show that Sigmar1 is a therapeutic target for methamphetamine-associated cardiomyopathy and defined the molecular mechanisms using autopsy samples of human hearts, and a mouse model of "binge and crash" methamphetamine administration. Sigmar1 expression is significantly decreased in the hearts of human methamphetamine users and those of "binge and crash" methamphetamine-treated mice. The hearts of methamphetamine users also show signs of cardiomyopathy, including cellular injury, fibrosis, and enlargement of the heart. In addition, mice expose to "binge and crash" methamphetamine develop cardiac hypertrophy, fibrotic remodeling, and mitochondrial dysfunction leading to contractile dysfunction. Methamphetamine treatment inhibits Sigmar1, resulting in inactivation of the cAMP response element-binding protein (CREB), decreased expression of mitochondrial fission 1 protein (FIS1), and ultimately alteration of mitochondrial dynamics and function. Therefore, Sigmar1 is a viable therapeutic agent for protection against methamphetamine-associated cardiomyopathy.

Methamphetamine induces cardiomyopathy by Sigmar1 inhibition-dependent impairment of mitochondrial dynamics and function.

Pulmonary microvascular endothelial cells (PMVECs) uniquely express an α-subtype of voltage-gated T-type Ca channel. We have previously revealed that the α channel functions as a background Ca entry pathway that is critical for the cell proliferation, migration, and angiogenic potential of PMVECs, a novel function attributed to the coupling between α-mediated Ca entry and constitutive Akt phosphorylation and activation. Despite this significance, mechanism(s) that link the α-mediated Ca entry to Akt phosphorylation remain incompletely understood. In this study, we demonstrate that Ca/calmodulin-dependent protein kinase (CaMK) 4 serves as a downstream effector of the α-mediated Ca entry to promote the angiogenic potential of PMVECs. Notably, CaMK2 and CaMK4 are both expressed in PMVECs. Pharmacological blockade or genetic knockdown of the α channel led to a significant reduction in the phosphorylation level of CaMK4 but not the phosphorylation level of CaMK2. Pharmacological inhibition as well as genetic knockdown of CaMK4 significantly decreased cell proliferation, migration, and network formation capacity in PMVECs. However, CaMK4 inhibition or knockdown did not alter Akt phosphorylation status in PMVECs, indicating that α/Ca/CaMK4 is independent of the α/Ca/Akt pathway in sustaining the cells' angiogenic potential. Altogether, these findings suggest a novel α-CaMK4 signaling complex that regulates the Ca-dominated angiogenic potential in PMVECs.

CaMK4 is a downstream effector of the α T-type calcium channel to determine the angiogenic potential of pulmonary microvascular endothelial cells.

Sigma 1 receptor (Sigmar1) is a widely expressed, multitasking molecular chaperone protein that plays functional roles in several cellular processes. Mutations in the Sigmar1 gene are associated with several distal neuropathies with strong manifestation in skeletal muscle dysfunction with phenotypes like muscle wasting and atrophy. However, the physiological function of Sigmar1 in skeletal muscle remains unknown. Herein, the physiological role of Sigmar1 in skeletal muscle structure and function in gastrocnemius, quadriceps, soleus, extensor digitorum longus, and tibialis anterior muscles was determined. Quantification of myofiber cross-sectional area showed altered myofiber size distribution and changes in myofiber type in the skeletal muscle of the Sigmar1 mice. Interestingly, ultrastructural analysis by transmission electron microscopy showed the presence of abnormal mitochondria, and immunostaining showed derangements in dystrophin localization in skeletal muscles from Sigmar1 mice. In addition, myopathy in Sigmar1 mice was associated with an increased number of central nuclei, increased collagen deposition, and fibrosis. Functional studies also showed reduced endurance and exercise capacity in the Sigmar1 mice without any changes in voluntary locomotion, markers for muscle denervation, and muscle atrophy. Overall, this study shows, for the first time, a potential physiological function of Sigmar1 in maintaining healthy skeletal muscle structure and function.

Molecular Characterization of Skeletal Muscle Dysfunction in Sigma 1 Receptor (Sigmar1) Knockout Mice.

Sigmar1 is a widely expressed molecular chaperone protein in mammalian cell systems. Accumulating research demonstrated the cardioprotective roles of pharmacologic Sigmar1 activation by ligands in preclinical rodent models of cardiac injury. Extensive biochemical and immuno-electron microscopic research demonstrated Sigmar1's sub-cellular localization largely depends on cell and organ types. Despite comprehensive studies, Sigmar1's direct molecular role in cardiomyocytes remains elusive. In the present study, we determined Sigmar1's subcellular localization, transmembrane topology, and function using complementary microscopy, biochemical, and functional assays in cardiomyocytes. Quantum dots in transmission electron microscopy showed Sigmar1 labeled quantum dots on the mitochondrial membranes, lysosomes, and sarcoplasmic reticulum-mitochondrial interface. Subcellular fractionation of heart cell lysates confirmed Sigmar1's localization in purified mitochondria fraction and lysosome fraction. Immunocytochemistry confirmed Sigmar1 colocalization with mitochondrial proteins in isolated adult mouse cardiomyocytes. Sigmar1's mitochondrial localization was further confirmed by Sigmar1 colocalization with Mito-Tracker in isolated mouse heart mitochondria. A series of biochemical experiments, including alkaline extraction and proteinase K treatment of purified heart mitochondria, demonstrated Sigmar1 as an integral mitochondrial membrane protein. Sigmar1's structural requirement for mitochondrial localization was determined by expressing FLAG-tagged Sigmar1 fragments in cells. Full-length Sigmar1 and Sigmar1's C terminal-deletion fragments were able to localize to the mitochondrial membrane, whereas N-terminal deletion fragment was unable to incorporate into the mitochondria. Finally, functional assays using extracellular flux analyzer and high-resolution respirometry showed Sigmar1 siRNA knockdown significantly altered mitochondrial respiration in cardiomyocytes. Overall, we found that Sigmar1 localizes to mitochondrial membranes and is indispensable for maintaining mitochondrial respiratory homeostasis in cardiomyocytes.

Judy King

Department of Pathology and Translational Pathobiology

Louisiana State University Health Sciences Center-Shreveport

Visualizing Subcellular Localization of a Protein in the Heart Using Quantum Dots-Mediated Immuno-Labeling Followed by Transmission Electron Microscopy