Novel large-particle FACS purification of adult ventricular myocytes reveals accumulation of myosin and actin disproportionate to cell size and proteome in normal post-weaning development

Rationale It is unknown if every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), beta-1, beta-2, beta-3, alpha-1A, and alpha-1B. The beta-1 and beta-2 are thought to be the dominant myocyte ARs. Objective Quantify the 5 cardiac ARs in individual ventricular myocytes. Methods and Results We studied ventricular myocytes from wild type mice, mice with alpha-1A and alpha-1B knockin reporters, and beta-1 and beta-2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of ERK and phospholamban), and contraction. We found that the beta-1 and alpha-1B were present in all myocytes. The alpha-1A was present in 60%, with high levels in 20%. The beta-2 and beta-3 were detected in only about 5% of myocytes, mostly in different cells. In intact heart, 30% of total beta-ARs were beta-2 and 20% were beta-3, both mainly in nonmyocytes. Conclusion The dominant ventricular myocyte ARs present in all cells are the beta-1 and alpha-1B. The beta-2 and beta-3 are mostly absent in myocytes but are abundant in nonmyocytes. The alpha-1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with beta-1 and alpha-1B only; 60% that also have the alpha-1A; and 5% each that also have the beta-2 or beta-3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms.

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“…72 Accordingly, it will be important to develop new methods to phenotype individual myocytes and relate phenotypes to receptor expression. 73 …”

Section: Discussionmentioning

confidence: 99%

Adrenergic Receptors in Individual Ventricular Myocytes

Myagmar

Flynn

Cowley

et al. 2017

Circulation Research

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104

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“…Moreover, scRNA-seq libraries generated from CMs following FACS 10 or isolation on the Fluidigm C1 4 have been dominated by mitochondrial reads, potentially suggesting that the cells were damaged prior to library generation. While one group has previously reported on isolation of rod-shaped CMs by FACS 11,12 , this approach only worked with fixed cells, and required significant instrument and sort parameter customization that may preclude its use in other labs. One approach that has been successful in generating high quality scRNA-seq libraries from single CMs has been single cell picking by micropipette 13 .…”

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confidence: 99%

Large particle fluorescence-activated cell sorting enables high quality single cell RNA-sequencing and functional analysis of adult cardiomyocytes

Kannan¹,

Miyamoto²,

Lin³

et al. 2019

Preprint

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Rationale: Single cell RNA sequencing (scRNA-seq) has emerged as a powerful tool to profile the transcriptome at single cell resolution, enabling comprehensive analysis of cellular trajectories and heterogeneity during development and disease. However, the use of scRNA-seq remains limited in cardiac pathology owing to technical difficulties associated with the isolation of single adult cardiomyocytes (CMs). Objective:We investigated the capability of large-particle fluorescence-activated cell sorting (LP-FACS) for isolation of viable single adult CMs. Methods and Results:We found that LP-FACS readily outperforms conventional FACS for isolation of struturally competent CMs, including large CMs. Additionally, LP-FACS enables isolation of fluorescent CMs from mosaic models. Importantly, the sorted CMs allow generation of high-quality scRNA-seq libraries. Unlike CMs isolated via previously utilized fluidic or manual methods, LP-FACisolated CMs generate libraries exhibiting normal levels of mitochondrial transcripts. Moreover, LP-FACS isolated CMs remain functionally competent and can be studied for contractile properties. Conclusions:Our study enables high quality dissection of adult CM biology at single-cell resolution.To date, however, scRNA-seq of adult CMs has been limited owing to technical difficulties in the isolation of single CMs. Specialized techniques are required for dissociation of the heart without damaging cells, as CMs are highly sensitive to pH, buffer ionic concentrations, and mechanical disruption 7 . Moreover, adult CMs are large (approximately 125x25µm 8 ), rod-shaped cells whose structure precludes isolation by either fluorescence-activated cell sorting (FACS) or commercial single cell microfluidic platforms (such as the Fluidigm C1 or Chromium 10X) 2,3 . Previous studies that have attempted to isolate CMs using conventional FACS equipment have resulted in frequent clogging of the instrument 9 or generation and/of fragmented myocytes 10 . Moreover, scRNA-seq libraries generated from CMs following FACS 10 or isolation on the Fluidigm C1 4 have been dominated by mitochondrial reads, potentially suggesting that the cells were damaged prior to library generation. While one group has previously reported on isolation of rod-shaped CMs by FACS 11,12 , this approach only worked with fixed cells, and required significant instrument and sort parameter customization that may preclude its use in other labs. One approach that has been successful in generating high quality scRNA-seq libraries from single CMs has been single cell picking by micropipette 13 . However, this approach is technically challenging, low throughput, and time intensive, which may in turn lead to significant transcriptomic changes during isolation. In lieu of whole cell scRNA-seq, others have utilized single nuclear RNA-seq (snRNA-seq), which overcomes the challenges of isolating large CMs [14][15][16] . While this approach can broadly capture cellular heterogeneity, snRNA-seq inherently detects fewer molecules and may skew gene expressi...

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“…To study cellular mechanisms of normal cardiac function and pathology, high-quality isolated adult myocytes are essential. [ 1 , 2 ] Reports on cell-to-cell imbalances in gene expression[ 3 – 5 ], heterogeneous expression of sarcomeric proteins[ 6 – 10 ], and adrenergic receptors[ 11 ] in myocyte sub-populations underscore the importance of single-cell analysis to understand cardiac biology. More broadly, single-cell analysis of all individual cells present in a heart is of high impact[ 12 ] and on par with the goals of the NIH Human BioMolecular Atlas Program.…”

Section: Introductionmentioning

confidence: 99%

“…In cardiac biology, the use of FCM in adult ventricular myocytes (VMs) isn’t frequently implemented in experiments due to the biophysical challenges caused by large, elongated, and irregularly shaped VMs. [ 6 , 10 ] In 1979, Nash and colleagues[ 14 ] reported the first evaluation of VM size and shape using a Coulter Counter Model B and a Coulter-Telefunken focused-flow system both fitted with a 200μm aperture. They reported the impact of aligning rod-shaped VMs with flow direction along a confined path: “Unfortunately this instrument tends to become blocked rather easily when used with heart muscle cells.”[ 14 ]…”

Section: Introductionmentioning

confidence: 99%

“…VMs were sorted through an Epics Elite ESP flow cytometer fitted with a 100μm sort tip. In 2017[ 10 ], we showed that mouse VMs fixed with paraformaldehyde (PFA) for FACS could not be easily purified in a BD Influx sorter fitted with a standard 100μm nozzle due to clogging in the instrument, as reported by Nash and colleagues[ 14 ]. Ultimately, a new large-particle set up with a 200μm nozzle was needed to overcome the biophysical challenge of purifying this large cell type.…”

Section: Introductionmentioning

confidence: 99%

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A method to increase reproducibility in adult ventricular myocyte sizing and flow cytometry: Avoiding cell size bias in single cell preparations

et al. 2017

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RationaleFlow cytometry (FCM) of ventricular myocytes (VMs) is an emerging technology in adult cardiac research that is challenged by the wide variety of VM shapes and sizes. Cellular variability and cytometer flow cell size can affect cytometer performance. These two factors of variance limit assay validity and reproducibility across laboratories. Washing and filtering of ventricular cells in suspension are routinely done to prevent cell clumping and minimize data variability without the appropriate standardization. We hypothesize that washing and filtering arbitrarily biases towards sampling smaller VMs than what actually exist in the adult heart.ObjectiveTo determine the impact of washing and filtering on adult ventricular cells for cell sizing and FCM.Methods and resultsLeft ventricular cardiac cells in single-cell suspension were harvested from New Zealand White rabbits and fixed prior to analysis. Each ventricular sample was aliquoted before washing or filtering through a 40, 70, 100 or 200μm mesh. The outcomes of the study are VM volume by Coulter Multisizer and light-scatter signatures by FCM. Data are presented as mean±SD.Myocyte volumes without washing or filtering (NF) served as the “gold standard” within the sample and ranged from 11,017 to 46,926μm3. Filtering each animal sample through a 200μm mesh caused no variation in the post-filtration volume (1.01+0.01 fold vs. NF, n = 4 rabbits, p = 0.999) with an intra-assay coefficient of variation (%CV) of <5% for all 4 samples. Filtering each sample through a 40, 70 or 100μm mesh invariably reduced the post-filtration volume by 41±10%, 9.0±0.8% and 8.8±0.8% respectively (n = 4 rabbits, p<0.0001), and increased the %CV (18% to 1.3%). The high light-scatter signature by FCM, a simple parameter for the identification of ventricular myocytes, was measured after washing and filtering. Washing discarded VMs and filtering cells through a 40 or 100μm mesh reduced larger VM by 46% or 11% respectively (n = 6 from 2 rabbits, p<0.001).ConclusionWashing and filtering VM suspensions through meshes 100μm or less biases myocyte volumes to smaller sizes, excludes larger cells, and increases VM variability. These findings indicate that validity and reproducibility across laboratories can be compromised unless cell preparation is standardized. We propose no wash prior to fixation and a 200μm mesh for filtrations to provide a reproducible standard for VM studies using FCM.

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Novel large-particle FACS purification of adult ventricular myocytes reveals accumulation of myosin and actin disproportionate to cell size and proteome in normal post-weaning development

Cited by 8 publications

References 59 publications

Adrenergic Receptors in Individual Ventricular Myocytes

Adrenergic Receptors in Individual Ventricular Myocytes

Large particle fluorescence-activated cell sorting enables high quality single cell RNA-sequencing and functional analysis of adult cardiomyocytes

A method to increase reproducibility in adult ventricular myocyte sizing and flow cytometry: Avoiding cell size bias in single cell preparations

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