Abstract Objectives To determine the role of MYL4 regulation of lysosomal function and its disturbance in fibrotic atrial cardiomyopathy. Background We have previously demonstrated that the atrial-specific essential light chain protein MYL4 is required for atrial contractile, electrical, and structural integrity. MYL4 mutation/dysfunction leads to atrial fibrosis, standstill, and dysrhythmia. However, the underlying pathogenic mechanisms remain unclear. Methods and results Rats subjected to knock-in of a pathogenic MYL4 mutant (p.E11K) developed fibrotic atrial cardiomyopathy. Proteome analysis and single-cell RNA sequencing indicate enrichment of autophagy pathways in mutant-MYL4 atrial dysfunction. Immunofluorescence and electron microscopy revealed undegraded autophagic vesicles accumulated in MYL4^p.E11K rat atrium. Next, we identified that dysfunctional MYL4 protein impairs autophagy flux in vitro and in vivo. Cardiac lysosome positioning and mobility were regulated by MYL4 in cardiomyocytes, which affected lysosomal acidification and maturation of lysosomal cathepsins. We then examined the effects of MYL4 overexpression via adenoviral gene-transfer on atrial cardiomyopathy induced by MYL4 mutation: MYL4 protein overexpression attenuated atrial structural remodeling and autophagy dysfunction. Conclusions MYL4 regulates autophagic flux in atrial cardiomyocytes via lysosomal mobility. MYL4 overexpression attenuates MYL4 p.E11K induced fibrotic atrial cardiomyopathy, while correcting autophagy and lysosomal function. These results provide a molecular basis for MYL4-mutant induced fibrotic atrial cardiomyopathy and identify a potential biological-therapy approach for the treatment of atrial fibrosis. Keywords: Myosin light chain 4, Atrial cardiomyopathy, Autophagy, Lysosome List of highlights * • Mutation of Myosin light chain 4 (MYL4) leads to atrial fibrosis, standstill, and dysrhythmia. * • The loss-of-function MYL4 blocks autophagy flux in atrial cardiomyocytes, leading to accumulation of undegraded vesicles. * • MYL4 regulates lysosome positioning and mobility in atrial cardiomyocytes. * • MYL4 overexpression attenuates atrial fibrosis caused by MYL4 p.E11K, while correcting atrial autophagy function. Abbreviations ATG10 Autophagy Related 10 ATG8A Autophagy-related 8a BAX B-Cell Lymphoma 2–Associated X Protein BCL-2 B-Cell Lymphoma 2 HO Homozygote HSPA8 Heat Shock Protein Family A Member 8 LAMP-2A Lysosomal-Associated Membrane Protein 2 LC3B Microtubule Associated Protein 1 Light Chain 3 Beta MAP1B Microtubule Associated Protein 1B MU Mutation MYH6 Myosin Heavy Chain 6 MYL3 Myosin Light Chain 3 MYL4 Myosin Light Chain 4 NRAMs Neonatal Rat Atrial Cardiomyocytes NRCM Neonatal Rat Cardiomyocytes NRVMs Neonatal Rat Ventricular Cardiomyocytes p62 Sequestosome 1 PBS Phosphate Buffered Saline TUNEL Terminal Deoxynucleotidyl Transferase Dutp Nick End Labeling WT Wild Type 1. Background Atrial cardiomyopathy is a common condition involving alterations in atrial structure, architecture, contraction, and/or electrophysiology [[47]1]. Only a limited number of studies have investigated the mechanisms underlying atrial cardiomyopathy. Myl4 encodes atrial essential light chain protein, which is expressed almost exclusively in the atria of adults [[48]2]. We previously identified a myosin light chain 4 (MYL4) loss-of-function mutation in an atrial standstill/arrhythmia family and established a MYL4 p.E11K knock-in rat model (MYL4^p.E11K rats) that reproduces the clinical phenotype with atrial-specific cardiomyocyte apoptosis and fibrotic lesions [[49]3]. Myosins are essential cytoskeletal motor proteins that transport intracellular particles along actin filaments to various cellular locations [[50]4], which was involved in many physiologic processes. Myosin I regulate cellular autophagy function by trafficking lysosome in its maturation or fusion with autophagosomes [[51]5,[52]6]. All myosins contain several light chains. There are two types of light chains in the myosin chaperone, the essential light chain (ELC) and the regulatory light chain. MYL4 is an atrial-specific ELC that is necessary for ATPase activity of the myosin head and also for actin binding [[53]7], a process that directly determines motor function. Here, we aimed to evaluate the potential role of dysregulation of autophagy and lysosomal function in the atrial cardiomyopathy caused by MYL4 mutation. We also aimed to determine whether MYL4 gene-transfer can correct the autophagy/lysosomal dysfunction caused by MYL4 mutation, and whether this intervention can prevent the associated atrial fibrosis. 2. Methods 2.1. Animal models All animal procedures were performed in accordance with NIH guidelines (Guide for the Care and Use of Laboratory Animals) and approved by the Animal Care and Use Committees of Shanghai Tenth People's Hospital (NO.SHDSYY-2020-3334). MYL4^p.E11K rat strains were generated by CRISPR/Cas-mediated genome editing as reported previously [[54]3]. 2.2. Primary cultures of neonatal rat cardiomyocytes Isolation and culture of primary neonatal rat cardiomyocytes (NRCMs) were prepared from heart of 1- to 3-days old WT and MYL4^p.E11K rats. In brief, the separated atriums and ventricles were digested in 1% collagenase II. The supernatant was stopped by culture medium (20% FBS, 1% penicillin and streptomycin in complete high glucose DMEM medium) and filtered by 100-μm cell strainer. Then collected supernatant supernatants was pre-plated for 60 min to remove fibroblasts and endothelial cells. The residual supernatant with cardiomyocytes was centrifuged at 300×g for 8 min and replanted in collagen-coated dishes at 5% CO[2] and 37 °C for 24 h. Cells were treated with 30 mM ammonium chloride (NH[4]Cl) (Sigma, USA) for 12 h to inhibit endosome-lysosome system acidification. 2.3. Plasmid transfection and adenovirus infection The plasmid targeting MYL4 (hU6-MCS-CMV-GFP-SV40-Neomycin) and control plasmid (CON036) were purchased from GENECHEM Incorporation (China). NRCMs were seeded in 12-well plate at the density of 5 × 10^5 cells/ml and transfected with 1.6 μg DNA using lipofectamine 2000 (Thermo Fisher, USA) in OPTI-MEN (Thermo Fisher, USA) for 24 h. The adenovirus for rat mutated MYL4 (Ad-MU-MYL4), WT MYL4 (Ad-WT-MYL4) and empty vector (Ad-Vector) were purchased from GENECHEM Incorporation (China) and the construction details and infection of NRCMs were described previously [[55]3]. For localized virus delivery, 10 μl 1E+10 PFU/ml Ad-WT-MYL4 or Ad-Vector was delivered by intramyocardial injection in MYL4^p.E11K rats’ left atrium. Adenoviruses encoding mRFP-GFP-LC3 was purchased from Hanbio Technology Corporation (China). Cardiomyocytes were infected with the virus at MOI 600 according to the manufacturer's instruction. 10 μl Ad-mRFP-GFP-LC3 was injected in 1-week MYL4 rat left ventricle for investigation of autophagy flux in vivo. After 3 weeks, survived animals were sacrificed by isoflurane. 2.4. Histological analysis For histological analysis, tissues were sliced at 5 μm thickness sections. Modified Masson's Trichrome Stain Kit (Scy Teklaboratories, USA) was used to determine interstitial fibrosis, which can distinguish blue fibrosis and red muscle. All sections were scanned with digital microscopy (Olympus, Japan) equipped with a 20X objective lens for 15 to 20 images per sample. All images were acquired under same conditions. The automatic exposure and white balance were turned off. Images were analyzed with Image J software (version 1.48v; National Institutes of Health). The area of fibers in deconvoluted color images was measured with ‘Threshold’ tool. Blood vessels, perivascular tissue and epicardium were excluded when measuring fibrotic area for fibrosis quantification. For transmission electron microscopy observation, rat hearts were fixed in 2% glutaraldehyde and immersed in 2% osmium tetroxide and 1% aqueous uranyl acetate for 1 h. The sample was washed by a series of ethanol solution and incubated into propylene oxide and EMbed 812 mixtures for 1 h, followed by polymerization at 70 °C. After sliced at 80 nm sections, 5% uranyl acetate and Reynold's lead citrate were used to stain. Sections were observed by a 40–120 kV transmission electron microscope (Hitachi H600 Electron Microscope, Hitachi, Japan). At least 10 fields of each sample were analyzed. 2.5. Western blot analysis Rat atrium tissue lysate and whole cells were prepared by 1x cell lysis (Cell Signaling Technologies, USA) and 1x protease inhibitors (Cat. 04693159001; Roche Molecular Biochemicals, USA). The crude extracts were centrifuged at 13000×g, 4 °C for 10 min and the supernatant was collected. The insoluble sediment was resuspended in 8 mM guanidine hydrochloride in complete lysis. The protein concentration was measured by bicinchoninic acid protein assay and equal amounts of protein samples were separated on 12% polyacrylamide gels, transferred onto PVDF membrane. Membranes were blocked in 5% non-fat milk or 3% BSA at room temperature and incubated with primary antibodies overnight at 4 °C. The primary antibodies targeted against Caspase-3 (Cell Signaling Technology, 9662s), BCL-2 (Abcam, ab32124), BAX (Abcam, ab32503), Vinculin (Santa Cruz Biotechnology, sc-73614), LAMP-2A (Abcam, ab125068), LC3B (Abcam, ab192890), p62 (Abcam, ab109012), MYL4 (Abnova, HOOOO4635-M01), Cathepsin B (Abcam, ab214428) or Cathepsin L (Santa Cruz Biotechnology, sc-390367) at a 1:1000 dilution. After 1-h HRP-conjugated secondary antibodies incubation, bends were visualized using chemiluminescence (ECL, TANON, China) and viewed under Amersham Imager 600 system (GE Healthcare, USA). The relative intensity of each band was normalized to Vinculin. 2.6. Quantitative real-time PCR RNA was extracted by Trizol reagent (Invitrogen, USA) and purified RNA (100 ng) was reverse-transcribed using HiScript III RT SuperMix (Vazyme, China). The quantitative RT-PCR was performed on 2 μg cDNA product using FastStart Universal SYBR Green Master (Roche, USA) on a Roche Lightcycler. Information of primers is presented in [56]Supplementary Table 1. 2.7. Immunofluorescence and confocal microscopy Rat heart tissue section and cells were fixed in 4% (w/v) paraformaldehyde (PFA)/PBS for 15 min, followed by permeabilization with 0.2% Triton X‐100 for 10 min and block with 5% BSA for 30 min at room temperature. First antibodies targeted against LAMP2 (Santa Cruz Biotechnology, sc-20004), LC3B (Abcam, ab192890) or MYL4 (Invitrogen, PA5-84091) at a dilution in 1:100 were incubated at 4 °C overnight. Second antibodies were incubated for 1 h and cell nucleus was stained by DAPI for 15 min at room temperature. Fluorescence images were obtained using Nikon fluorescent microscope (Nikon, Japan). 2.8. Time-lapse live-cell imaging NRCMs were incubated with Hoechst 33342 (Beyotime, China) and Lyso-Tracker Green (Beyotime, China) at the concentrations of 1:2000 and 1:20000 respectively for 30 min and washed by HSBB solution for 3 min. Then NRCMs were incubated with completed culture medium and imaged using Nikon confocal microscope. The moving distance of lysosome was measured by Image J software. 2.9. Live-cell lysosomal acidification NRCM lysosome acidification was detected by fluorescence intensity using Lyso- Sensor Green (Yeasen, China) with 30 min incubation. This dye is concentrated in acid cell component, of which fluorescence intensity is higher in lower pH component. Each group was captured for 10 to 15 fields using Nikon confocal microscope. All the images were analyzed by Image J software to measure lysosome acidification. The average pH value in knock-down group was normalized to the average value of control group. 2.10. Single-cell RNA sequencing database analysis The scRNA-seq data were downloaded from the Gene Expression Omnibus database (GEO; [57]GSE128908). Seurat's (version 3.1.1 in R[[58]8]) standard process was conducted to analyze the sequencing results. In brief, genes expressed in less than 10 cells and cells with fewer than 200 unique molecular identifiers (UMIs) or mitochondrial gene expression exceeding 60% were excluded. Clustering of filtered cell data set was identified by highly variable genes on their mean expression from 0.05 to 10 and dispersion between 1.5 and 20. The clusters were presented by t-distributed stochastic neighbor embedding (t-SNE) using dimensionality reduction. The Shared Nearest Neighbor (SNN) graph was constructed with 50 nearest neighbors and 20 dimensions of PCs as input. Clusters were identified using the above graph with resolution parameter of 0.6. Differential gene expression among clusters used MAST test in Seurat and gene expression over 25% cells was considered as significant difference. P value < 0.05 and |log2foldchange| > 1 was set as the threshold for significantly differential expression. GO enrichment and KEGG pathway enrichment analysis of DEGs were respectively performed using R based on the hypergeometric distribution. 2.11. 4D label-free quantitative proteomics NRAMs (1X10 [[59]6]) obtained from neonatal WT and MYL4 rat atrium were thawed in SDT (4%SDS，100 mM Tris-HCl, 1 mM DTT, pH7.6) buffer on ice. The amount of protein was quantified with the BCA Protein Assay Kit (Bio-Rad, USA). 20 μg of protein for each sample were mixed with 5X loading buffer respectively and boiled for 5 min. The proteins were separated on 12.5% SDS-PAGE gel (constant current 14 mA, 90 min). Protein bands were visualized by Coomassie Blue R-250 staining. The lysate was digested by trypsin according to filter-aided sample preparation (FASP) procedure and then desalted on C18 Cartridges (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 ml, Sigma). Digested peptides were concentrated by vacuum centrifugation and reconstituted in 40 μl of 0.1% (v/v) formic acid. The LC-MS/MS analysis was operated on the timsTOF Pro mass spectrometer (Bruker) in positive ion mode with 100–1700 m/z and 0.6 to 1.6 1/k0, coupled to Nanoelute (Bruker Daltonics) for 60 min. The peptides were loaded onto a reverse phase trap column (Thermo Scientific Acclaim PepMap100, 100 μm*2 cm, nanoViper C18) connected to the C18-reversed phase analytical column (Thermo Scientific Easy Column, 10 cm long, 75 μm inner diameter, 3 μm resin) in buffer A (0.1% Formic acid) and separated with a linear gradient of buffer B (84% acetonitrile and 0.1% Formic acid) at a flow rate of 300 nl/min controlled by IntelliFlow technology. The mass spectrometer was operated in positive ion mode. The mass spectrometer collected ion mobility MS spectra over a mass range of m/z 100–1700 and 1/k0 of 0.6–1.6, and then performed 10 cycles of PASEF MS/MS with a target intensity of 1.5 k and a threshold of 2500. Active exclusion was enabled with a release time of 0.4 min. MaxQuant 1.6.14.0 software is used for identification and quantitation analysis of MS raw data. The original data files were available on the iProX database ([60]https://www.iprox.cn/page/home.html; IPX0005144000). The studied proteins were blasted against the online Kyoto Encyclopedia of Genes and Genomes (KEGG) database ([61]http://geneontology.org/) to retrieve their KEGG orthology identifications and were subsequently mapped to pathways in KEGG. Enrichment analysis were performed by the Fisher’ exact test and p-value was adjusted by Benjamini- Hochberg correction for multiple testing. Both functional categories and pathways with p-values below 0.05 was considered statistically significant. 2.12. Statistical analysis Data were analyzed using the package for social sciences (SPSS) for Windows 10. Numerical variables with a normal distribution are presented as the mean ± SEM. For intergroup comparisons of numerical variables, an independent sample t-test and Mann-Whitney U test were used. Bonferroni corrections were used to adjust the p-value to reduce type I errors in multiple comparisons. A two-tailed p < 0.05 was considered to be statistically significant. 3. Results 3.1. Autophagy-associated pathways are identified by proteome analysis of the atria of MYL4^p.E11K rats We first separated atrial cardiomyocytes and confirmed the presence of atrial cardiomyocyte markers ([62]Supplemental Figs. 1A–D). To identify proteins enriched in the atria of MYL4^p.E11K rats, we performed 4D label-free quantification proteome analysis on WT and MYL4^p.E11K rat atrial cardiomyocytes N = 3). On average, 3896 proteins per MYL4 and 3877 proteins per WT sample were detected ([63]Fig. 1A). A total of 203 proteins were upregulated and 158 proteins were downregulated in the MYL4^p.E11K group. These differentially expressed proteins were significantly enriched in cardiac muscle contraction-, metabolism-, and protein degradation-related pathways ([64]Fig. 1B). Interestingly, the MYL4 rat samples demonstrated downregulation of autophagy, a canonical protein degradation pathway ([65]Fig. 1C). The levels of certain autophagy-related proteins MAP1A, CTSB, MAP1S and RAB1B were significantly decreased ([66]Fig. 1D). Fig. 1. [67]Fig. 1 [68]Open in a new tab Autophagy-associated pathways are identified via proteome analysis of the atria of MYL4^p.E11Krats. (A) Venn diagram showing the protein expression overlap in WT and MYL4 rat atrial cardiomyocytes. (B) Relative expression differences between WT and MYL4 groups as illustrated by a Treemap of condensed gene ontology (GO) terms. The sizes of boxes corresponded to the number of significant terms associated with the GO category. (C) Heat map analysis of expression pattern of cardiomyopathy and autophagy associated genes. (D) Differently expressed genes between WT and MYL4^p.E11K rats in the autophagy pathway. CTSB = cathepsin B; DAPI = 40,6-diamidino-2-phenylindole; FoxO = forkhead box class O; HO = homozygote; RAB1B = member RAS oncogene family; MAP1A = microtubule associated protein 1A; MAP1S = microtubule associated protein 1S; WT = wild type. Two-tailed Student's t-test is used to compare two groups; Data are presented as mean ± SEM. *p < 0.05, **p < 0.001; n = 3. We re-analyzed the single-cell RNA sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO; [69]GSE128908) that recorded scRNA-seq results of MYL4 ± and MYL4−/− hESC-atrial cells as well as MYL4 +/+ controls [[70]9]. After filtration and quality control, 14,861 digested single cells were subjected to subsequent analysis. We then defined 11 cell clusters by cluster analysis and evaluated gene expression differences among MYH6+ hESC-atrial cells ([71]Fig. 2A–C). The mutant cell lines were significantly enriched for the dilated cardiomyopathy pathway and the autophagy-related pathway according to scoring of individual cells for pathway activities [[72]10] ([73]Fig. 2D–F). [74]Fig. 2G indicates that MYL4−/− cells highly expressed the autophagy-related genes Atg8a, Map1b, Atg10, and Hspa8. The altered gene expression was further confirmed by RT-qPCR ([75]Fig. 2H) in samples collected from MYL4^p.E11K rat atrium. Taken together, these findings suggest that MYL4 dysfunction strongly affects atrial cardiomyocytes autophagy related gene expression. Fig. 2. [76]Fig. 2 [77]Open in a new tab Upregulation of autophagy and dilated cardiomyopathy pathway in MYL4 mutants. (A, B) t-SNE plot of single-cell clusters in combined WT and mutant populations. The mutant unique cluster (#2,7,4) is circled in red. (C) t-SNE overlay of atrial lineage maker MYH6 expressed in cardiomyocyte fraction. (D) Dotplot heatmap showed GO biological process terms enriched in cluster marker genes. (E, F) t-SNE plot of MYH6 high group in combined WT and mutant populations and selected transcription factor network activity AUC score cluster distributions. (G) Violin plots of selected top differentially expressed genes between WT and mutant lines in autophagy pathway. (H) Quantitative RT-PCR analysis of top different genes expression in MYL4 p.E11K rat atrium tissue. Atg8a = autophagy-related protein 8a; Atg10 = autophagy related 10; Hspa8 = heat shock protein family A member 8; Map1b = microtubule associated protein 1B. Other abbreviations are the same as those in [78]Fig. 1. Data are presented as mean ± SEM. Two-tailed Student's t-test is used to compare two groups; *p < 0.05, **p < 0.001; for MYL4+/+ group, n = 4, for MYL4 ± and MYL4−/−group, n = 8. (For interpretation of the references to color in this figure legend, the