Aging reduces the number and function of L-type calcium channels in the membrane of cardiac pacemaker cells

Figures

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  Figure 1.. Identification of the sinoatrial node and pacemaker cells. A. Schematic representation of the position of the sinoatrial node (SAN) pacemaker in the heart, located between the right atrium (RA) and the superior and inferior vena cava (SVC, IVC). B. Volumetric reconstruction of a cleared SAN explant immuno-stained against HCN4 with identified regions for the head and tail. (Right) Magnification of five different regions showing cellular organization inside the tissue. C. Schematic representation of the procedure to dissect and isolate single pacemaker cells. D, E. Pictures of single cells stained against HCN4 and RyR2 after isolation. Panel D shows three representative pacemaker cells positive for HCN4. Panel E shows three representative cells negative for HCN4.

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  Figure 2.. Aging slows down the action potential firing rate and the diastolic depolarization of pacemaker cells. A. Representative recordings of spontaneous action potential from young (black) and old (orange) pacemaker cells. B. Schematic of two pacemaker action potentials depicting the parameters analyzed (CL: cycle length, APD: action potential duration, RD: repolarization duration, APA: action potential amplitude, MDP: maximum diastolic depolarization, DD: diastolic depolarization, EDD: Early DD, LDD: Late DD. C. Fold change of the action potential parameters analyzed in old pacemaker cells relative to young. The vertical dotted line represents the young population. Purple bars highlight parameters that were significantly different from the young. D. Comparison of the firing rate from young and old pacemaker cells. E. Comparison of the standard deviation (SD) of the cycle length from young and old pacemaker cells. F. Comparison of the DD duration from young and old pacemaker cells. G. Representative traces of the diastolic depolarization phase from a young and old pacemaker cell to compare the Early DD (green) and Late DD (blue) slopes. Adjusted lines were used to calculate the…

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  Figure 3.. L-type calcium current density is reduced in old pacemaker cells. A. Representative recordings of the total calcium current (ICa TOTAL, black traces) and the nifedipine-resistant component (ICaT, light and dark purple traces) from young and old pacemaker cells. The shaded region of the current corresponds to the nifedipine-sensitive component (ICaL, grey for young and orange for old). B. Comparison of calcium current density with age for both L- and T-type. C. Percentages of ICaL and ICaT relative to total calcium currents observed in each age group. Note that in this plot, total calcium current in old cells is also shown as 100%, but it is only 50% of the current observed in young cells. D. Comparison of the current-voltage relationship for ICaL from young and old pacemaker cells. E. Normalized conductance (G) - voltage relationship for ICaL from young and old pacemaker cells. In all the scattered plots, bars represent the mean, and error bars the SEM. Statistical comparisons used a two-tailed t-test comparing a population of n = 16 cells, N = 4 mice for the young group and n = 16 cells, N = 4 mice for the old.

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  Figure 4.. Aging does not lead to cell hypertrophy. A. Pictures of heart explants showing the pacemaker region stained against HCN4 in young (left) and old (right) mice. B. Comparison of cell width from HCN4-positive cells measured from tissue explants. C. Comparison of cell length from HCN4-positive cells measured from tissue explants. Measurements in B - D were performed from three tissues in each age, and the total number of cells was 37 in the young and 39 in the old. D. Pictures of isolated pacemaker cells from young (top) or old (bottom) mice stained against HCN4. Comparison of short axis (E), long axis (F), and cell area (G) from isolated HCN4-positive cells. Measurements in panels E-G are from 53 pacemaker cells in young and 54 pacemaker cells in old. H. Comparison of the capacitance of young and old pacemaker cells. Sample numbers were n = 22 cells, N = 4 mice for the young group, and n = 21 cells, N = 4 mice for the old.

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  Figure 5.. Aging does not affect the total expression of L-type calcium channels but reduces their density near the plasma membrane. A. Representative western blots for CaV1.2 and CaV1.3 channels and total protein stains from pacemaker tissue explants from young and old mice. B. Fold change of CaV1.2 and CaV1.3 channel total expression in old animals relative to young. Expression was normalized to total protein and each data point represents an animal. Statistical comparisons used a two-tail Mann-Whitney test. C-F. Representative AiryScan high-resolution images of the footprint of young and old pacemaker cells labeled against CaV1.2 (magenta) or CaV1.3 (orange). Insets are 5 μm by 5 μm footprint regions from each of the cells shown to the left. G. Comparison of CaV1.2 and CaV1.3 particle density between young (CaV1.2, n = 22, N = 3; CaV1.3, n = 16, N = 3) and old (CaV1.2, n= 33, N = 3; CaV1.3, n = 23, N = 3) cells. Statistical comparisons used a two-tail t-test.

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  Figure 6.. Aging reduces L-type calcium channel clustering. A and B. Representative super-resolution images of CaV1.2 and CaV1.3 channels in young and old pacemaker cells. Panels to the right of each image are zoom in. C. Average cluster area for CaV1.2 and CaV1.3 channel clusters in young and old pacemaker cells. D. Comparison of CaV1.2 and CaV1.3 cluster density between young and old cells. E and F. Comparison of the frequency distributions of the area of CaV1.2 and CaV1.3 channel clusters between young and old cells. In all the scattered plots bars represent the mean and error bars the SEM. Statistical comparisons used a two-tail t-test comparing a population of n = 8 cells, N = 3 mice for CaV1.2 young; n = 12 cells, N = 4 mice for CaV1.3 young; n = 6 cells, N = 3 mice for CaV1.2 old; and n = 8 cells, N = 3 mice for CaV1.3 old.

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  Figure 7.. Aging reduces the number of active channels at the plasma membrane and their open probability. A. Representative single-channel recordings from young (black) and old (orange) pacemaker cells. Dotted lines represent number of channels opening (1–7 channels), from the baseline closed state based on a unitary current of 0.7 pA. B. Comparison of the channel open probability (NPΟ) between young and old pacemaker cells calculated from 50 voltage sweeps per cell. Statistical comparisons used a two-tail t-test comparing a population of n = 9 cells, N = 3 mice for the young group and n = 8 cells, N = 3 mice for the old group.

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  Figure 8.. Increasing L-type calcium channel open probability restores normal pacemaking rates in old cells. A. Representative recordings of action potentials from young and old pacemaker cells before (black) and after (red) application of 500 nM Bay K8644. B. Comparison of the effect of Bay K on the firing rate of young and old pacemaker cells. C. Fold change of the action potential parameters analyzed in old pacemaker cells relative to young in control (black) and Bay K (red). Vertical dotted lines represent the young population. Purple bars highlight parameters that were significantly different in control conditions but reverted by Bay K. D. Representative overlapped magnification of the diastolic depolarization in the absence (control) and presence of Bay K. Straight lines illustrate how the kinetics of the Early DD (green) and Late DD (blue) phases were calculated from young (dotted) and old (solid) traces. E. Comparison of the effect of Bay K (red) on the duration of the diastolic depolarization (DD) relative to control (black) in young and old pacemaker cells. F,G. Comparison of the slopes of the tangential lines for Early DD (F) and Late DD (G) between young and old pacemaker cells…