DOI: 10.5061/dryad.pvmcvdnn9
title: Data from: Functional remodelling of perinuclear mitochondria alters nucleoplasmic Ca2+ signalling in heart failure

description: Mitochondrial dysfunction in cardiomyocytes is a hallmark of heart failure
 (HF) development. Although initial studies recognized the importance of
 different mitochondrial subpopulations, there is a striking lack of direct
 comparison of intrafibrillar (IF) vs. perinuclear (PN) mitochondria during
 the development of HF. Here, we use multiple approaches to examine the
 morphology and functional properties of IF vs. PN mitochondria in pressure
 overload-induced cardiac remodeling in mice, and in non-failing and
 failing human cardiomyocytes. We demonstrate that PN mitochondria from
 failing cardiomyocytes are more susceptible to depolarization of
 mitochondrial membrane potential (ΔΨm), ROS generation and impairment in
 Ca2+ uptake compared to IF mitochondria at baseline and under
 physiological stress protocol. We also demonstrated, for the first time,
 that under normal conditions PN mitochondrial Ca2+ uptake shapes
 nucleoplasmic Ca2+ transients (CaTs) and limits nucleoplasmic Ca2+
 loading. Loss of PN mitochondrial Ca2+ buffering capacity translates into
 increased nucleoplasmic CaTs and may explain disproportionate rise in
 nucleoplasmic [Ca2+] in failing cardiomyocytes at increased stimulation
 frequencies. Therefore, a previously unidentified benefit of restoring the
 mitochondrial Ca2+ uptake may be normalization of nuclear Ca2+ signaling
 and alleviation of altered excitation-transcription, which could be an
 important therapeutic approach to prevent adverse cardiac remodeling.
description: The following datasets are available: 
 A custom MATLAB script for semi-automated analysis of
 cytoplasmic, nucleoplasmic, or subcellular Ca²⁺ transients in electrically
 stimulated cardiomyocytes. A
 step-by-step user guide to facilitate the use of the script.
 Supplemental materials, including expanded
 Materials and Methods, Supplemental Table 1, and Supplemental Figure
 1. Data supporting Figures 1–6,
 including full-resolution images used in the figures and GraphPad files
 with individual data values and corresponding statistical
 analyses.
description: # Data from: Functional remodelling of perinuclear mitochondria alters
 nucleoplasmic Ca2+ signalling in heart failure
 [https://doi.org/10.5061/dryad.pvmcvdnn9](https://doi.org/10.5061/dryad.pvmcvdnn9) ## Description of the data The following files are available: 1. A custom MATLAB script for semi-automated analysis of cytoplasmic, nucleoplasmic, or subcellular Ca²⁺ transients in electrically stimulated cardiomyocytes (file format: MATLAB code; file name: *Script for semi-automated CaTs analyses*). 2. A step-by-step user guide to facilitate the use of the script (file format: docx; file name: *Detailed Instructions for Using the MATLAB Script*). 3. Data supporting Figures 1–6 (each in individual folder named: *Figure 1*, *Figure 2*..., *Figure 6*), including full-resolution images used in the figures (file format: jpg, folder name: *Full resolution images*) and GraphPad files with individual data values and corresponding statistical analyses (file format: GraphPad Prism Project; file name: *Figure 1b*, *Figure 1d*, ..., *Figure 6*). ## File structure **Main Folder Name: *Perinuclear mitochondria in cardiomyocytes*** Content (6 sub-folders, 1 docx file and 1 MATLAB code):  **1) Sub-folder 1: *Figure 1*** * Sub-sub-folder: *Full resolution images* * *Figure 1a.jpg*: Original full size electron microscopy (EM) image of intrafibrillar (IF) and perinuclear (PN) mitochondria in representative left ventricular section of a healthy mouse heart. * *Figure 1ci - DAPI.jpg*: Original full size fluorescence image of a live adult mouse cardiomyocyte stained with DAPI to locate cardiomyocyte nuclei. * *Figure 1ci - Mitotracker.jpg*: Original full size fluorescence image of a live adult mouse cardiomyocyte stained with Mitotracker to locate cardiomyocyte mitochondria. * *Figure 1ci - Overlay_Mitotracker&DAPI.jpg*: Overlay of original full size fluorescence images of a live adult mouse cardiomyocyte stained with DAPI and Mitotracker to locate cardiomyocyte nuclei and mitochondria, respectively. * *Figure 1ciii*: Original full size EM image of PN mitochondria accumulated in the space between two nuclei of a typically binucleated mouse ventricular myocyte. * File Name: *Figure 1b-Perimeter and Aspect Ratio.prism - *GraphPad Prism file of individual data points and statistical analyses corresponding to Figure 1b. * File Name: *Figure 1d-Sham vs TAC.prism - *GraphPad Prism file of individual data points and statistical analyses corresponding to Figure 1d. **2) Sub-folder 2: *Figure 2*** * Sub-sub-folder: *Full resolution images* * *Sham.jpg*: Original full size fluorescence image of a live ventricular cardiomyocyte isolated from control (sham) mice and stained with TMRM. * *TAC 7W.jpg*: Original full size fluorescence image of a live ventricular cardiomyocyte isolated from mice 7 weeks after the transverse aortic constriction (TAC) and stained with TMRM. - File Name: *Figure 2b-2d-Sham vs TAC.prism - *GraphPad Prism file of individual data points and statistical analyses corresponding to Figure 2b and 2d. - File Name: *Notes for Figure 2.docx - *Docx file summarizing important points for statistical analyses corresponding to data in Figure 2. **3) Sub-folder 3: *Figure 3*** * Sub-sub-folder: *Full resolution images* * *Figure 3a - sham_baseline.jpg*: Original full size fluorescence image of a live ventricular cardiomyocyte isolated from control (sham) mice and stained with TMRM at rest. * *Figure 3a - TAC 1W_baseline.jpg*: Original full size fluorescence image of a live ventricular cardiomyocyte isolated from mice 1 weeks after TAC and stained with TMRM at rest. * *Figure 3a - TAC 7W_baseline.jpg*: Original full size fluorescence image of a live ventricular cardiomyocyte isolated from mice 7 weeks after TAC and stained with TMRM at rest. * *Figure 3c - Sham_cell1_1Hz_5min-1*: Original full size fluorescence image of a live ventricular cardiomyocyte isolated from control (sham) mice and stained with TMRM in response to 5-10 min of low-frequency stimulation (1 Hz). * *Figure 3c - Sham_cell1_5Hz_5min-1*: Original full size fluorescence image of a live ventricular cardiomyocyte isolated from control (sham) mice and stained with TMRM in response to 5 min of high-frequency stimulation (5 Hz). * *Figure 3c - Sham_cell1_5Hz_10min-1*: Original full size fluorescence image of a live ventricular cardiomyocyte isolated from control (sham) mice and stained with TMRM in response to 10 min of high-frequency stimulation (5 Hz). * *Figure 3c - Figure 3c - TAC_cell8_1Hz_5min-1*: Original full size fluorescence image of a live ventricular cardiomyocyte isolated from mice 7 weeks after TAC and stained with TMRM in response to 5-10 min of low-frequency stimulation (1 Hz). * *Figure 3c - Figure 3c - TAC_cell8_5Hz_5min-1*: Original full size fluorescence image of a live ventricular cardiomyocyte isolated from mice 7 weeks after TAC and stained with TMRM in response to 5 min of high-frequency stimulation (5 Hz). * *Figure 3c - Figure 3c - TAC_cell8_5Hz_10min-1*: Original full size fluorescence image of a live ventricular cardiomyocyte isolated from mice 7 weeks after TAC and stained with TMRM in response to 10 min of high-frequency stimulation (5 Hz). * *Figure 3e - failing_human myocyte.jpg*: Original full size fluorescence image of a live ventricular cardiomyocyte isolated from a failing human heart and stained with TMRM at rest. * *Figure 3e - NF_human myocyte.jpg*: Original full size fluorescence image of a live ventricular cardiomyocyte isolated from a non-failing human heart and stained with TMRM at rest. * File Name: *Figure 3b-Membrane Potential_baseline.prism - *GraphPad Prism file of individual data points and statistical analyses corresponding to Figure 3b. * File Name: *Figure 3d-Membrane Potential_pacing.prism - *GraphPad Prism file of individual data points and statistical analyses corresponding to Figure 3d. * File Name: *Figure 3f-Membrane Potential_human.prism - *GraphPad Prism file of individual data points and statistical analyses corresponding to Figure 3f. **4) Sub-folder 4: *Figure 4*** * Sub-sub-folder: *Full resolution images* * *Sham_CellROX_NT_0.5Hz_cell3-1.jpg*: Original full size fluorescence image of ROS production in response to 10 min of low-frequency stimulation (0.5 Hz) in cardiomyocytes isolated from a sham mouse heart, detected by CellROX® Deep Red reagent staining. * *Sham_CellROX_NT_5Hz_cell5-1.jpg*: Original full size fluorescence image of ROS production in response to 10 min of high-frequency stimulation (5 Hz) in cardiomyocytes isolated from a sham mouse heart, detected by CellROX® Deep Red reagent staining.  * *TAC_CellRox_NT_0.5Hz_cell1-1.jpg*: Original full size fluorescence image of ROS production in response to 10 min of low-frequency stimulation (0.5 Hz) in cardiomyocytes isolated from a mouse heart 7 weeks after TAC, detected by CellROX® Deep Red reagent staining. * *TAC_CellRox_NT_5Hz_cell6-1.jpg*: Original full size fluorescence image of ROS production in response to 10 min of high-frequency stimulation (5 Hz) in cardiomyocytes isolated from a mouse heart 7 weeks after TAC, detected by CellROX® Deep Red reagent staining. - File Name: *Figure 4b-CellRox.prism - *GraphPad Prism file of individual data points and statistical analyses corresponding to Figure 4b. - File Name: *Figure 4c-4d-Mito Ca uptake.prism - *GraphPad Prism file of individual data points and statistical analyses corresponding to Figure 4c and 4d. - File Name: *Figure 4e-Mito Ca uptake rate.prism - *GraphPad Prism file of individual data points and statistical analyses corresponding to Figure 4e. **5) Sub-folder 5: *Figure 5*** * Sub-sub-folder: *Full resolution images* * *Figure 1a-Fluo-4_NT.jpg*: Original full size line scans of calcium transients (CaTs) visualized by Fluo-4 staining in a healthy murine cardiomyocyte before the application of the mitochondrial Ca2+ uptake inhibitor Ru360, stimulated at 2.5 Hz. * *Figure 1a-Fluo-4_Ru360.jpg*: Original full size line scans of calcium transients (CaTs) visualized by Fluo-4 staining in a healthy murine cardiomyocyte upon  the application of the mitochondrial Ca2+ uptake inhibitor Ru360, stimulated at 2.5 Hz. * *Figure 1a-TMRM-4_NT.jpg*: Original full size line scans of mitochondria visualized by TMRM staining in a healthy murine cardiomyocyte before the application of the mitochondrial Ca2+ uptake inhibitor Ru360, stimulated at 2.5 Hz. * *Figure 1a-TMRM-4_Ru360.jpg*: Original full size line scans of mitochondria visualized by TMRM staining in a healthy murine cardiomyocyte upon the application of the mitochondrial Ca2+ uptake inhibitor Ru360, stimulated at 2.5 Hz. * *Figure 1di-Fluo-4_NT_human.jpg*: Original full size line scans of calcium transients (CaTs) visualized by Fluo-4 staining in a non-failing cardiomyocyte, stimulated at 0.5 Hz. * *Figure 1dii-Fluo-4_Ru360_human.jpg*: Original full size line scans of mitochondria visualized by TMRM staining in a non-failing cardiomyocyte, stimulated at 0.5 Hz. - Figure 5c-Ru360*.prism - *GraphPad Prism file of individual data points and statistical analyses corresponding to Figure 5c. **6) Sub-folder 6: *Figure 6*** * File Name: *Figure 6_CaTs Sham vs TAC.prism - *GraphPad Prism file of individual data points and statistical analyses corresponding to Figure 6. * File Name: *Notes for Figure 6.docx - *Docx file summarizing important points for statistical analyses corresponding to data in Figure 6. **7) File: *Detailed Instructions for Using the MATLAB Script.docx*** A Docx file with the step-by-step user guide to facilitate the use of the MATLAB script. **8) File: *Script for semi-automated CaTs analyses.m*** A custom MATLAB script for semi-automated analysis of cytoplasmic, nucleoplasmic, or subcellular Ca²⁺ transients in electrically stimulated cardiomyocytes. Please see the detailed explanation below, in the section "Code/software". # Code/software This MATLAB script included in this dataset is designed to analyze electrically stimulated Ca2+ transients (CaTs) in various sub-compartments of cardiomyocytes. It begins by reading time-point data from an Excel file, isolating fully recorded CaTs, and averaging and smoothing the signals. Key parameters, including baseline, peak, and decay times, are calculated for each CaT, and the results are saved to a new Excel file. To use the script, the user imports data by specifying the Excel file name, then uses the "Run Section" function to visualize the sequence of the recorded CaTs and align individual CaTs for averaging and improving the signal-to-noise ratio. After achieving the best alignment, the user specifies an output file name and runs the remaining sections to generate the analyzed data file. Additional customization options are available, allowing the user to add or adjust parameters based on specific research questions. All steps of the process are detailed in the file titled *Detailed Instructions for Using the MATLAB Script*, which is included in the dataset.