RGS4 controls Gαi3-mediated regulation of Bcl-2 phosphorylation on TGN38-containing intracellular membranes.

Intracellular pools of the heterotrimeric G-protein α-subunit Gαi3 (encoded by GNAI3) have been shown to promote growth factor signaling, while at the same time inhibiting the activation of JNK and autophagic signaling following nutrient starvation. The precise molecular mechanisms linking Gαi3 to both stress and growth factor signaling remain poorly understood. Importantly, JNK-mediated phosphorylation of Bcl-2 was previously found to activate autophagic signaling following nutrient deprivation. Our data shows that activated Gαi3 decreases Bcl-2 phosphorylation, whereas inhibitors of Gαi3, such as RGS4 and AGS3 (also known as GPSM1), markedly increase the levels of phosphorylated Bcl-2. Manipulation of the palmitoylation status and intracellular localization of RGS4 suggests that Gαi3 modulates phosphorylated Bcl-2 levels and autophagic signaling from discreet TGN38 (also known as TGOLN2)-labeled vesicle pools. Consistent with an important role for these molecules in normal tissue responses to nutrient deprivation, increased Gαi signaling within nutrient-starved adrenal glands from RGS4-knockout mice resulted in a dramatic abrogation of autophagic flux, compared to wild-type tissues. Together, these data suggest that the activity of Gαi3 and RGS4 from discreet TGN38-labeled vesicle pools are critical regulators of autophagic signaling that act via their ability to modulate phosphorylation of Bcl-2.

Membrane proteomic profiling of the heart: past, present, and future.

Cardiovascular diseases remain the most rapidly rising contributing factor of all-cause mortality and the leading cause of inpatient hospitalization worldwide, with costs exceeding $30 billion annually in North America. Cell surface and membrane-associated proteins play an important role in cardiomyocyte biology and are involved in the pathogenesis of many human heart diseases. In cardiomyocytes, membrane proteins serve as critical signaling receptors, Ca2+ cycling regulators, and electrical propagation regulators, all functioning in concert to maintain spontaneous and synchronous contractions of cardiomyocytes. Membrane proteins are excellent pharmaceutical targets due to their uniquely exposed position within the cell. Perturbations in cardiac membrane protein localization and function have been implicated in the progression and pathogenesis of many heart diseases. However, previous attempts at profiling the cardiac membrane proteome have yielded limited results due to poor technological developments for isolating hydrophobic, low-abundance membrane proteins. Comprehensive mapping and characterization of the cardiac membrane proteome thereby remains incomplete. This review will focus on recent advances in mapping the cardiac membrane proteome and the role of novel cardiac membrane proteins in the healthy and the diseased heart.

Three-dimensional imaging reveals endo(sarco)plasmic reticulum-containing invaginations within the nucleoplasm of muscle.

The mammalian nucleus has invaginations from the cytoplasm, termed nucleoplasmic reticulum (NR). With increased resolution of cellular imaging, progress has been made in understanding the formation and function of NR. In fact, nucleoplasmic Ca2+ homeostasis has been implicated in the regulation of gene expression, DNA repair, and cell death. However, the majority of studies focus on cross-sectional or single-plane analyses of NR invaginations, providing an incomplete assessment of its distribution and content. Here, we provided advanced imaging and three-dimensional reconstructive analyses characterizing the molecular constituents of nuclear invaginations in the nucleoplasm in HEK293 cells, murine C2C12 muscle cells, and cardiac myocytes. We demonstrated the presence of critical Ca2+ regulatory channels, including sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a), stromal interaction molecule 1 (STIM1), and Ca2+ release-activated Ca2+ channel protein 1 (ORAI1), in the nucleoplasm in isolated primary mouse cardiomyocytes. We have shown for the first time the presence of STIM1 and ORAI1 in the nucleoplasm, suggesting the presence of store-operated calcium entry (SOCE) mechanism in nucleoplasmic Ca2+ regulation. These results show that nucleoplasmic invaginations contain continuous endoplasmic reticulum components, mitochondria, and intact nuclear membranes, highlighting the extremely detailed and complex nature of this organellar structure.

Impact of COVID-19 on hospital screening, diagnosis and treatment activities among prostate and colorectal cancer patients in Canada.

BACKGROUND: Suspension of cancer screening and treatment programs were instituted to preserve medical resources and protect vulnerable populations. This research aims to investigate the implications of COVID-19 on cancer management and clinical outcomes for patients with prostate and colorectal cancer in Canada. METHODS: We examined hospital cancer screening, diagnosis, treatment, length of stay, and mortality data among prostate and colorectal cancer patients between April 2017 and March 2021. Baseline trends were established with data between April 2017 and March 2020 for comparison with data collected between April 2020 and March 2021. Scenario analyses were performed to assess the incremental capacity requirements needed to restore hospital cancer care capacities to the pre-pandemic levels. RESULTS: For prostate cancer, A 12% decrease in diagnoses and 5.3% decrease in treatment activities were observed during COVID-19 between April 2020 and March 2021. Similarly, a 43% reduction in colonoscopies, 11% decrease in diagnoses and 10% decrease in treatment activities were observed for colorectal cancers. An estimated 1,438 prostate and 2,494 colorectal cancer cases were undiagnosed, resulting in a total of 620 and 1,487 unperformed treatment activities for prostate and colorectal cancers, respectively, across nine provinces in Canada. To clear the backlogs of unperformed treatment procedures will require an estimated 3%-6% monthly capacity increase over the next 6 months. INTERPRETATION: A concerted effort from all stakeholders is required to immediately ameliorate the backlogs of cancer detection and treatment activities. Mitigation measures should be implemented to minimize future interruptions to cancer care in Canada.

Towards understanding the role of Receptor Expression Enhancing Protein 5 (REEP5) in cardiac muscle and beyond.

The sarco-endoplasmic reticulum (SR/ER) is the largest membrane-bound organelle in eukaryotic cells and plays important roles in essential cellular processes, and in development and progression of many cardiac diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. In a recently published study led by Lee et al. (Nat Commun 11(1):965), we reported a cardiac enriched SR/ER membrane protein REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes resulted in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca2+ cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants showed sensitized cardiac dysfunction to heart failure induction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrated cardiac dysfunction with dilated cardiac chambers, increased cardiac fibrosis, and reduced ejection fraction. These results demonstrate the critical role of REEP5 in SR/ER organization and function.

Bioinformatic analysis of membrane and associated proteins in murine cardiomyocytes and human myocardium.

In the current study we examined several proteomic- and RNA-Seq-based datasets of cardiac-enriched, cell-surface and membrane-associated proteins in human fetal and mouse neonatal ventricular cardiomyocytes. By integrating available microarray and tissue expression profiles with MGI phenotypic analysis, we identified 173 membrane-associated proteins that are cardiac-enriched, conserved amongst eukaryotic species, and have not yet been linked to a 'cardiac' Phenotype-Ontology. To highlight the utility of this dataset, we selected several proteins to investigate more carefully, including FAM162A, MCT1, and COX20, to show cardiac enrichment, subcellular distribution and expression patterns in disease. We performed three-dimensional confocal imaging analysis to validate subcellular localization and expression in adult mouse ventricular cardiomyocytes. FAM162A, MCT1, and COX20 were expressed differentially at the transcriptomic and proteomic levels in multiple models of mouse and human heart diseases and may represent potential diagnostic and therapeutic targets for human dilated and ischemic cardiomyopathies. Altogether, we believe this comprehensive cardiomyocyte membrane proteome dataset will prove instrumental to future investigations aimed at characterizing heart disease markers and/or therapeutic targets for heart failure.

Functional culture and in vitro genetic and small-molecule manipulation of adult mouse cardiomyocytes.

Primary adult cardiomyocyte (aCM) represent the mature form of myocytes found in the adult heart. However, culture of aCMs in particular is challenged by poor survival and loss of phenotype, rendering extended in vitro experiments unfeasible. Here, we establish murine aCM culture methods that enhance survival and maintain sarcomeric structure and Ca2+ cycling to enable physiologically relevant contractile force measurements. We also demonstrate genetic and small-molecule manipulations that probe mechanisms underlying myocyte functional performance. Together, these refinements to aCM culture present a toolbox with which to advance our understanding of myocardial physiology.

Mapping signalling perturbations in myocardial fibrosis via the integrative phosphoproteomic profiling of tissue from diverse sources.

Study of the molecular basis of myocardial fibrosis is hampered by limited access to tissues from human patients and by confounding variables associated with sample accessibility, collection, processing and storage. Here, we report an integrative strategy based on mass spectrometry for the phosphoproteomic profiling of normal and fibrotic cardiac tissue obtained from surgical explants from patients with hypertrophic cardiomyopathy, from a transaortic-constriction mouse model of cardiac hypertrophy and fibrosis, and from a heart-on-a-chip model of cardiac fibrosis. We used the integrative approach to map the relative abundance of thousands of proteins, phosphoproteins and phosphorylation sites specific to each tissue source, to identify key signalling pathways driving fibrosis and to screen for anti-fibrotic compounds targeting glycogen synthase kinase 3, which has a consistent role as a key mediator of fibrosis in all three types of tissue specimen. The integrative disease-modelling strategy may reveal new insights into mechanisms of cardiac disease and serve as a test bed for drug screening.

REEP5 depletion causes sarco-endoplasmic reticulum vacuolization and cardiac functional defects.

The sarco-endoplasmic reticulum (SR/ER) plays an important role in the development and progression of many heart diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. Here, we report a cardiac enriched, SR/ER membrane protein, REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes results in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca2+ cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants show sensitized cardiac dysfunction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrates cardiac dysfunction. These results demonstrate the critical role of REEP5 in SR/ER organization and function as well as normal heart function and development.

Modeling cardiac complexity: Advancements in myocardial models and analytical techniques for physiological investigation and therapeutic development in vitro.

Cardiomyopathies, heart failure, and arrhythmias or conduction blockages impact millions of patients worldwide and are associated with marked increases in sudden cardiac death, decline in the quality of life, and the induction of secondary pathologies. These pathologies stem from dysfunction in the contractile or conductive properties of the cardiomyocyte, which as a result is a focus of fundamental investigation, drug discovery and therapeutic development, and tissue engineering. All of these foci require in vitro myocardial models and experimental techniques to probe the physiological functions of the cardiomyocyte. In this review, we provide a detailed exploration of different cell models, disease modeling strategies, and tissue constructs used from basic to translational research. Furthermore, we highlight recent advancements in imaging, electrophysiology, metabolic measurements, and mechanical and contractile characterization modalities that are advancing our understanding of cardiomyocyte physiology. With this review, we aim to both provide a biological framework for engineers contributing to the field and demonstrate the technical basis and limitations underlying physiological measurement modalities for biologists attempting to take advantage of these state-of-the-art techniques.

Nanoscale reorganization of sarcoplasmic reticulum in pressure-overload cardiac hypertrophy visualized by dSTORM.

Pathological cardiac hypertrophy is a debilitating condition characterized by deleterious thickening of the myocardium, dysregulated Ca2+ signaling within cardiomyocytes, and contractile dysfunction. Importantly, the nanoscale organization, localization, and patterns of expression of critical Ca2+ handling regulators including dihydropyridine receptor (DHPR), ryanodine receptor 2 (RyR2), phospholamban (PLN), and sarco/endoplasmic reticulum Ca2+-ATPase 2A (SERCA2A) remain poorly understood, especially during pathological hypertrophy disease progression. In the current study, we induced cardiac pathological hypertrophy via transverse aortic constriction (TAC) on 8-week-old CD1 mice, followed by isolation of cardiac ventricular myocytes. dSTORM super-resolution imaging was then used to visualize proteins at nanoscale resolution at two time points and we quantified changes in protein cluster properties using Voronoi tessellation and 2D Fast Fourier Transform analyses. We showed a decrease in the density of DHPR and RyR2 clusters with pressure-overload cardiac hypertrophy and an increase in the density of SERCA2A protein clusters. PLN protein clusters decreased in density in 2-week TAC but returned to sham levels by 4-week TAC. Furthermore, 2D-FFT analysis revealed changes in molecular organization during pathological hypertrophy, with DHPR and RyR2 becoming dispersed while both SERCA2A and PLN sequestered into dense clusters. Our work reveals molecular adaptations that occur in critical SR proteins at a single molecule during pressure overload-induced cardiomyopathy. Nanoscale alterations in protein localization and patterns of expression of crucial SR proteins within the cardiomyocyte provided insights into the pathogenesis of cardiac hypertrophy, and specific evidence that cardiomyocytes undergo significant structural remodeling during the progression of pathological hypertrophy.