An expanded proteome of cardiac t-tubules

Cheah, Jenice X.; Nieuwenhuis, Tim O.; Halushka, Marc K.

doi:10.1016/j.carpath.2019.05.001

Cited by 5 publications

(7 citation statements)

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“…Most fibers had between 500-700 proteins identified. As we have stated repeatedly, IHC in the HPA is subject to false positive staining from shared epitopes (20, 24–26). It also incurs false negative staining for failed antibodies or antibodies with staining parameters designed for other tissues.…”

Section: Discussionmentioning

confidence: 82%

“…The HPA is a comprehensive repository of IHC stained tissue microarrays for numerous tissues, including skeletal muscle (35, 36). The HPASubC tool can rapidly and agnostically interrogate images of the HPA to characterize specific staining patterns in organs (20, 24, 26). HPASubC v1.2.4 was used to download 50,351 skeletal muscle tissue microarray images covering 10,301 unique proteins from the HPA website (v18).…”

Section: Methodsmentioning

confidence: 99%

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Proteogenomic single cell analysis of skeletal muscle myocytes

Fomchenko

Verma

Kannan

et al. 2020

Preprint

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39 40 Skeletal muscle myocytes have evolved into slow and fast-twitch types. These types are 41 functionally distinct as a result of differential gene and protein expression. However, an 42 understanding of the complexity of gene and protein variation between myofibers is unknown. 43We performed deep, whole cell, single cell RNA-seq on intact and fragments of skeletal 44 myocytes from the mouse flexor digitorum brevis muscle. We compared the genomic expression 45 data of 171 of these cells with two human proteomic datasets. The first was a spatial proteomics 46 survey of mosaic patterns of protein expression utilizing the Human Protein Atlas (HPA) and the 47 HPASubC tool. The second was a mass-spectrometry (MS) derived proteomic dataset of single 48 human muscle fibers. Immunohistochemistry and RNA-ISH were used to understand variable 49 expression. scRNA-seq identified three distinct clusters of myocytes (a slow/fast 2A cluster and 50 two fast 2X clusters). Utilizing 1,605 mosaic patterned proteins from visual proteomics, and 596 51 differentially expressed proteins by MS methods, we explore this fast 2X division. Only 36 52 genes/proteins had variable expression across all three studies, of which nine are newly described 53 as variable between fast/slow twitch myofibers. An additional 414 genes/proteins were identified 54 as variable by two methods. Immunohistochemistry and RNA-ISH generally validated variable 55 expression across methods presumably due to species-related differences. In this first integrated 56 proteogenomic analysis of mature skeletal muscle myocytes we confirm the main fiber types and 57 greatly expand the known repertoire of twitch-type specific genes/proteins. We also demonstrate 58 the importance of integrating genomic and proteomic datasets. 59 60

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Section: Discussionmentioning

confidence: 82%

Section: Methodsmentioning

confidence: 99%

Proteogenomic single cell analysis of skeletal muscle myocytes

Fomchenko

Verma

Kannan

et al. 2020

Preprint

Self Cite

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“…A number of limitations should be recognized. As we have stated repeatedly, IHC in the HPA is subject to false-positive staining from shared epitopes, although the requirement of variable, mosaic expression within a single core of tissues reduces this cause of error. − This method also has false negatives for any failed antibodies or antibodies with poor staining as a result of methodologic parameters designed for other tissues. Further, some proteins that are likely mosaic are missing in HPA.…”

Section: Discussionmentioning

confidence: 99%

“…HPA is a comprehensive repository of IHC-stained tissue microarrays for numerous tissues, including skeletal muscles. , The HPASubC tool can rapidly and agnostically interrogate images of HPA to characterize specific staining patterns in organs. ,, HPASubC v1.2.4 was used to download 49,600 unique skeletal muscle tissue microarray images covering 12,620 unique proteins from the HPA website (v18). The images were individually reviewed using HPASubC by K.M.F.…”

Section: Methodsmentioning

confidence: 99%

Spatial Proteomic Approach to Characterize Skeletal Muscle Myofibers

et al. 2020

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Skeletal muscle myofibers have differential protein expression resulting in functionally distinct slow- and fast-twitch types. While certain protein classes are well-characterized, the depth of all proteins involved in this process is unknown. We utilized the Human Protein Atlas (HPA) and the HPASubC tool to classify mosaic expression patterns of staining across 49,600 unique tissue microarray (TMA) images using a visual proteomic approach. We identified 2164 proteins with potential mosaic expression, of which 1605 were categorized as “likely” or “real.” This list included both well-known fiber-type-specific and novel proteins. A comparison of the 1605 mosaic proteins with a mass spectrometry (MS)-derived proteomic dataset of single human muscle fibers led to the assignment of 111 proteins to fiber types. We additionally used a multiplexed immunohistochemistry approach, a multiplexed RNA-ISH approach, and STRING v11 to further assign or suggest fiber types of newly characterized mosaic proteins. This visual proteomic analysis of mature skeletal muscle myofibers greatly expands the known repertoire of twitch-type-specific proteins.

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“…Several recent studies have expanded the role of Jph2 as a key player in dyad assembly by interacting directly with LTCCs [75], and additionally, acting in a transcriptional capacity under conditions of stress and disease [76]. Cheah et al [77] characterized the T-tubule subproteome using immunohistochemical images from the Human Protein Atlas cardiac tissue microarrays, and identified 78 novel putative T-tubule proteins and generated a single interactome map. These revelations and the potential role of thyroid hormones in maintaining dyadic couplings in cardiomyocytes will advance therapeutic approaches in the treatment of heart disease.…”

Section: T3 Regulates Tt and Sr Structural And Functional Genesmentioning

confidence: 99%

Triiodothyronine maintains cardiac transverse-tubule structure and function

Gilani

Wang

Muncan

et al. 2021

Journal of Molecular and Cellular Cardiology

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Subclinical hypothyroidism and low T3 syndrome are commonly associated with an increased risk of cardiovascular disease (CVD) and mortality. We examined effects of T3 on T-tubule (TT) structures, Ca 2+ mobilization and contractility, and clustering of dyadic proteins.Thyroid hormone (TH) deficiency was induced in adult female rats by propyl-thiouracil (PTU; 0.025%) treatment for 8 weeks. Rats were then randomized to continued PTU or triiodo-L-thyronine (T3; 10 μg/kg/d) treatment for 2 weeks (PTU + T3). After in vivo echocardiographic and hemodynamic recordings, cardiomyocytes (CM) were isolated to record Ca 2+ transients and contractility. TT organization was assessed by confocal microscopy, and STORM images were captured to measure ryanodine receptor (RyR2) cluster number and size, and L-type Ca 2+ channel (LTCC, Ca v 1.2) co-localization. Expressed genes including two integral TT proteins, junctophilin-2 (Jph-2) and bridging integrator-1 (BIN1), were analyzed in left ventricular (LV) tissues and cultured CM using qPCR and RNA sequencing. The T3 dosage used normalized serum T3, and reversed adverse effects of TH deficiency on in vivo measures of cardiac function. Recordings of isolated CM indicated that T3 increased rates of Ca 2+ release and re-uptake, resulting in increased velocities of sarcomere shortening and re-lengthening. TT periodicity was significantly decreased, with reduced transverse tubules but increased longitudinal tubules in TH-deficient CMs and LV tissue, and these structures were normalized by T3 treatment. Analysis of STORM data of PTU myocytes showed decreased RyR2 cluster numbers and RyR localizations within each cluster without significant changes in Ca v 1.2 localizations within RyR clusters. T3 treatment normalized RyR2 cluster size and number. qPCR and RNAseq analyses of LV and cultured CM showed that Jph2 expression was T3-responsive, and its increase with treatment may explain improved TT organization and RyR-LTCC coupling.

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An expanded proteome of cardiac t-tubules

Cited by 5 publications

References 50 publications

Proteogenomic single cell analysis of skeletal muscle myocytes

Proteogenomic single cell analysis of skeletal muscle myocytes

Spatial Proteomic Approach to Characterize Skeletal Muscle Myofibers

Triiodothyronine maintains cardiac transverse-tubule structure and function

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