Dataset supporting the manuscript:
Vivas O, Baudot M, Madden R, Pinon-Teal WL, Hunt MS, Choi S, Pournejati R, Flores VA, Santana LF, Moreno CM. Aging Reduces the Number and Function of L-type Calcium Channels in the Membrane of Cardiac Pacemaker Cells. Circulation Research. Submission ID: CIRCRES/2025/327894DR1.
Corresponding author: Claudia M. Moreno (morenocm@uw.edu)

Related publication DOI: [to be added upon publication]

License

This dataset is released under the Creative Commons Zero (CC0) waiver.

Summary

This dataset contains the raw numerical data underlying the figures of the manuscript. The study quantifies age-associated changes in the electrical activity, calcium handling, protein expression, and nanoscale organization of sinoatrial node (SAN) pacemaker cells. Experiments were performed on pacemaker cells isolated from young (4–6 months) and old (24–30 months) male C57BL/6 mice. Measurements span whole-cell electrophysiology (action potentials, calcium currents, single-channel recordings), calcium imaging, confocal and super-resolution (STORM) microscopy of ion channel distributions, and Western blot quantification of pacemaker proteins.

How to open these files

All data files are plain UTF-8 encoded comma-separated values (CSV). They can be opened with:

• Any text editor (Notepad, TextEdit, VS Code, etc.)
• Microsoft Excel, Numbers, LibreOffice Calc (open directly)
• R: read.csv("filename.csv")
• Python (pandas): pandas.read_csv("filename.csv")
• MATLAB: readtable("filename.csv")

Each CSV contains two columns. Headers are in row 1; data begins in row 2.

Empty cells in any column indicate no observation at that row position (the two columns are padded to equal length for display purposes only). They do not represent missing data.

Software used for analysis

Statistical analyses were performed in:

• GraphPad Prism 10 — used for unpaired t-tests, nested t-tests, and two-tailed Mann-Whitney tests.
• R version 4.3 or later with the lme4 package — used for the linear mixed effects models in Figure 9 panels B, F, G, and H. Model formula: value ~ age * condition + (1 | mouse/cell_id). Post-hoc comparisons used Sidak correction for four pre-specified contrasts.
• ImageJ (NIH) — used to quantify Western blot band intensities as area under the curve normalized to total protein.

No custom analysis scripts are required to reproduce the comparisons reported in the manuscript. Raw values in the CSV files can be entered directly into Prism (or an equivalent package) to reproduce the statistical tests.

Abbreviations and symbols

Experimental context (common to all files)

All experiments were performed on pacemaker cells or sinoatrial node tissue from male C57BL/6 mice. Two age groups were used throughout the study:

• Young: 4–6 months
• Old: 24–30 months

Electrophysiology recordings were performed at 32–34 °C. Full experimental details (cell isolation, patch-clamp, calcium imaging, STORM, Western blot protocols) are provided in the Methods section of the manuscript.

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File descriptions

The dataset is organized by manuscript figure. Each CSV contains two columns; the comparison column labels are indicated below.

Figure 2 — Spontaneous action potential parameters in young vs old pacemaker cells

Perforated-patch current-clamp recordings of spontaneous action potentials from isolated pacemaker cells.

Sample sizes: Young n = 28 cells, N = 5 mice / Old n = 18 cells, N = 5 mice. Columns Young and Old are unpaired.

Statistical test: nested t-test (cells nested within animals), GraphPad Prism 10.

Figure 3 — Whole-cell calcium currents

Whole-cell voltage-clamp recordings in response to 20-ms depolarizing pulses to –20 mV from a holding potential of –80 mV. The L-type component (I_CaL) was isolated by subtracting the nifedipine-resistant current (10 μM nifedipine) from the total calcium current; the nifedipine-resistant component represents the T-type current (I_CaT).

Sample sizes: Young n = 16 cells, N = 4 mice / Old n = 16 cells, N = 4 mice.

Statistical test: two separate nested t-tests (one for L-type, one for T-type); these are two independent hypotheses, not a multigroup comparison.

Figure 4 — Pacemaker cell size

Three complementary approaches test whether aging affects pacemaker cell size: (1) cell dimensions measured from HCN4-immunostained whole-mount tissue explants; (2) cell dimensions measured from single isolated pacemaker cells; and (3) whole-cell capacitance as an electrophysiological measure of cell surface area.

Statistical test: nested t-test (cells nested within animals or tissues), GraphPad Prism 10.

Figure 5 — Calcium clearance machinery (NCX1) and SR calcium load

Three complementary approaches test whether the age-associated reduction in L-type current reflects changes in calcium clearance or SR load: (1) NCX1 total protein expression by Western blot; (2) NCX1 surface density by AiryScan confocal imaging; and (3) SR calcium content estimated from caffeine-induced calcium transients.

Panel A — NCX1 total expression (Western blot)

Each data point is one animal; values are fold change relative to the young group.

Statistical test (Panel A): two-tailed Mann-Whitney test (N < 10 per group).

Immunofluorescence quantification of NCX1 on AiryScan confocal images

Caffeine-induced SR calcium transient

Cells were loaded with Cal-520 and exposed to a localized 20 mM caffeine puff in calcium-free external solution.

Statistical test: nested t-test (cells nested within animals), GraphPad Prism 10.

Figure 6 — Caᵥ1.2 and Caᵥ1.3 total expression and surface density

Figure 6 tests whether aging affects the total protein expression (Panel A, Western blot) and the plasma membrane surface density (Panels D–K, AiryScan footprint imaging) of Ca_V1.2 and Ca_V1.3 L-type calcium channels. Ca_V1.2 and Ca_V1.3 were measured in independent experimental preparations and are not paired comparisons on the same biological unit.

Panel A — Total protein expression (Western blot)

Each data point is one animal; values are fold change relative to the young group.

Statistical test (Panel A): two independent two-tailed Mann-Whitney tests (one for Ca_V1.2, one for Ca_V1.3). N < 10 per group.

Panels D–K — Surface density by AiryScan confocal imaging

Channel surface density at the basal (footprint) plane of isolated pacemaker cells.

Statistical test (Panels D, E, J, K): nested t-test (cells nested within animals), GraphPad Prism 10.

Figure 7 — Nanoscale organization of Ca_V1.2 and Ca_V1.3 clusters (STORM)

Super-resolution microscopy (Stochastic Optical Reconstruction Microscopy, STORM) in TIRF mode was used to quantify channel clusters at the basal region (footprint plane) of isolated pacemaker cells, with a Z-axis penetration depth of approximately 100 nm. Ca_V1.2 and Ca_V1.3 were imaged in separate cells from independent preparations — they are two independent comparisons, not multiple comparisons on the same biological unit.

Statistical test: nested t-test (cells nested within animals), GraphPad Prism 10.

Figure 8 — Single-channel activity (cell-attached patch)

Cell-attached patch-clamp recordings. N·Po (number of active channels × open probability) was calculated from 50 voltage sweeps per patch.

Sample sizes: Young n = 9 cells, N = 3 mice / Old n = 11 cells, N = 3 mice.

Statistical test: nested t-test (cells nested within animals), GraphPad Prism 10.

Figure 9 — L-type calcium channel agonist (Bay K 8644) rescue experiment

Paired measurements before and after application of the L-type calcium channel agonist Bay K 8644 in the same cells. For panels B, F, G, and H, the paired repeated-measures structure was analyzed with a linear mixed effects model accounting for both the within-cell paired design and the nesting of cells within mice. Panels K, M, and O report pre-computed fold-change values (post/pre Bay K 8644) for each cell.

Sample sizes (panels B, F, G, H): Young n = 14 cells, N = 3 mice / Old n = 11 cells, N = 3 mice.
Panel E (DD duration): Young n = 14 / Old n = 10 (one old cell had no measurable DD and is excluded).

Statistical test (panels B, F, G, H): linear mixed effects model fitted with the lme4 package in R (value ~ age * condition + (1|mouse/cell_id)), with Sidak correction for four pre-specified contrasts: (1) young-control vs young + Bay K 8644; (2) old-control vs old + Bay K 8644; (3) young-control vs old-control; (4) young + Bay K 8644 vs old + Bay K 8644.
Statistical test (panels K, M, O): nested t-test comparing fold changes between young and old.

Figure 10 — Caveolin-3 and calcium channel surface organization

This figure combines Western blot quantification of caveolin-3 abundance (panel B), confocal immunofluorescence of caveolin-3 at the cell edge (panel D), and STORM imaging of Caᵥ1.2 and Caᵥ1.3 cluster density and area after caveolar disruption with methyl-β-cyclodextrin (MβCD, 5 mM, 30 min; panels F and G).

Statistical tests:

• Panel 10B: two-tailed Mann-Whitney test (N < 10 per group at the animal level; Old group deviated from normality, Shapiro-Wilk p = 0.014).
• Panels 10D, 10F, 10G: nested t-test (cells nested within animals), GraphPad Prism 10.