Arrhythmogenesis in the aged heart following ischaemia-reperfusion: role of transient receptor potential vanilloid 4

Abstract

Aims: Cardiomyocyte Ca2+ homoeostasis is altered with ageing and predisposes the heart to Ca2+ intolerance and arrhythmia. Transient receptor potential vanilloid 4 (TRPV4) is an osmotically activated cation channel with expression in cardiomyocytes of the aged heart. The objective of this study was to examine the role of TRPV4 in Ca2+ handling and arrhythmogenesis following ischaemia-reperfusion (I/R), a pathological scenario associated with osmotic stress.

Methods and results: Cardiomyocyte membrane potential was monitored prior to and following I/R in Langendorff-perfused hearts of Aged (19-28 months) male and female C57BL/6 mice ± TRPV4 inhibition (1 μM HC067047, HC). Diastolic resting membrane potential was similar between Aged and Aged HC at baseline, but following I/R Aged exhibited depolarized diastolic membrane potential vs. Aged HC. The effects of TRPV4 on cardiomyocyte Ca2+ signalling following I/R were examined in isolated hearts of Aged cardiac-specific GCaMP6f mice (±HC) using high-speed confocal fluorescence microscopy, with cardiomyocytes of Aged exhibiting an increased incidence of pro-arrhythmic Ca2+ signalling vs. Aged HC. In the isolated cell environment, cardiomyocytes of Aged responded to sustained hypoosmotic stress (250mOsm) with an increase in Ca2+ transient amplitude (fluo-4) and higher incidence of pro-arrhythmic diastolic Ca2+ signals vs. Aged HC. Intracardiac electrocardiogram measurements in isolated hearts following I/R revealed an increased arrhythmia incidence, an accelerated time to ventricular arrhythmia, and increased arrhythmia score in Aged vs. Aged HC. Aged exhibited depolarized resting membrane potential, increased pro-arrhythmic diastolic Ca2+ signalling, and greater incidence of arrhythmia when compared with Young (3-5 months).

Conclusion: TRPV4 contributes to pro-arrhythmic cardiomyocyte Ca2+ signalling, electrophysiological abnormalities, and ventricular arrhythmia in the aged mouse heart.

Keywords: Arrhythmia; Calcium; Excitation-contraction coupling; GCaMP; Ischemia–reperfusion.

Graphical abstract

Figure 1

TRPV4 contributes to membrane depolarization following ischaemia–reperfusion in aged hearts. (A) Example left ventricular AP from cardiomyocytes of an isolated perfused Aged heart (left) and Aged heart pre-treated with the TRPV4 antagonist HC067047 (Aged HC, 1 µmol/L, right) under baseline conditions. (B) Summary data of cardiomyocyte resting membrane potential (RMP) of Aged (burgundy) and Aged HC (grey) under baseline conditions. (C) Frequency histograms of RMP with associated Gaussian fits of cardiomyocyte populations under baseline conditions in Aged (burgundy) and Aged HC (grey) hearts. (D) Example left ventricular APs from cardiomyocytes of an isolated perfused Aged (left) and Aged HC heart (right) following ischaemia–reperfusion (I/R). (E) Summary data of cardiomyocyte RMP of Aged (burgundy) and Aged HC (grey) following I/R. (F) Frequency histograms of RMP with associated Gaussian fits (Double Gaussian Aged; Single Gaussian Aged HC) of cardiomyocyte populations following I/R in Aged (burgundy) and Aged HC (grey). n = 52 cells from N = 6 animals Aged baseline; n = 47 cells from N = 5 animals Aged HC baseline; n = 122 cells from N = 6 animals Aged I/R; n = 108 cells from N = 5 animals Aged HC I/R. *P = 0.019 Aged vs. Aged HC, nested t test (E).

Figure 2

Cardiomyocyte action potential properties following ischaemia–reperfusion in aged hearts. (A) Linear regression (solid line) with 95% prediction bands (dashed lines) of AP amplitude vs. RMP following I/R of Aged (burgundy, left) Aged pre-treated with the TRPV4 antagonist HC067047 (Aged HC, 1 µmol/L, grey, centre). Overlay of linear regression (solid lines) and prediction bands (dashed lines) are shown at right for Aged (burgundy) and Aged HC (grey). (B) Linear regression (solid line) with 95% prediction bands (dashed lines) of AP upstroke velocity vs. RMP following I/R of Aged (burgundy, left) and Aged HC (grey, centre). Overlay of linear regression (solid lines) and prediction bands (dashed lines) are shown at right for Aged (burgundy) and Aged HC (grey). (C) Linear regression (solid line) with 95% prediction bands (dashed lines) of AP decay time (90–10%) vs. RMP following I/R of Aged (burgundy, left) and Aged HC (grey, centre) from −90 to −60 mV. Overlay of linear regression (solid lines) and prediction bands (dashed lines) are shown at right for Aged (burgundy) and Aged HC (grey). (D) Linear regression (solid line) with 95% prediction bands (dashed lines) of AP decay time (90 − 10%) vs. RMP following I/R of Aged (burgundy, left) and Aged HC (grey, centre) constrained from −60 to −35 mV. n = 178–219 cells from N = 5–6 animals Aged; n = 141–148 cells from N = 4 animals Aged HC (A–C). n = 41 cells from N = 6 animals Aged; n = 7 cells from N = 4 animals Aged HC (D). Note minimal points in Aged HC in this RMP range, with no data points between −50 and −35 mV. One Aged HC heart was excluded from regression analysis due to low rate (<2 Hz).

Figure 3

Cardiomyocyte Ca²⁺ signalling following ischaemia–reperfusion in aged hearts. (A) Schematic of MerCreMer × GCaMP6f double transgenic mice. When treated with tamoxifen ‘STOP’ sequence is excised leading to GCaMP6f expression in cardiomyocytes. Unfiltered and filtered light (460–480 nm excitation/500–540 emission) images of an isolated perfused Aged GCaMP6f heart shown at right. (B) Example high-speed two-dimensional confocal images of GCaMP6f fluorescence in sub-epicardial cardiomyocytes of an Aged GCaMP6f heart during diastole (upper) and peak systole (lower) under baseline conditions (left) and following I/R (right). Raw fluorescence profiles (8 bits) of baseline cells 1 and 2 (left) and I/R cells 3 and 4 (right) shown below images. Dashed lines indicate diastolic and systolic fluorescence values of a typical cell within the field, and illustrate heterogeneity in Aged following I/R (cells 3 and 4). (C) Summary super plots of Ca²⁺ transient amplitude (F/F₀, calculated per cell) in Aged and Aged pre-treated with TRPV4 antagonist HC067047 (Aged HC, 1 µmol/L) under baseline conditions (left) and following I/R (right). Individual cardiomyocyte F/F₀ values are presented (n = 155 Aged and n = 180 Aged HC baseline; n = 580 Aged and n = 513 Aged HC I/R) with sample mean ± SEM calculated per heart (N = 5 Aged, N = 3 Aged HC) and shown to right of individual cardiomyocyte scatter plots, with corresponding colour. Aged and Aged HC had equal variance under baseline conditions (F test P = 0.4, nested t test P = 0.47) but unequal variance following I/R (F test P < 0.001, nested t test P = 0.2). (D) Example high-speed (100 Hz) two-dimensional confocal images of GCaMP6f fluorescence in sub-epicardial cardiomyocytes of an Aged GCaMP6f heart during diastole (upper) and peak systole (lower) following I/R. Raw fluorescence profiles of stable cell (cell 1) and cell exhibiting spontaneous Ca²⁺ waves (cell 2) are shown below images. Dashed lines indicate diastolic and systolic fluorescence of cell 1 (left). Summary data of percent of total cells (79/1,658 Aged vs. 0/942 Aged HC) exhibiting Ca²⁺ wave or Ca²⁺ overload behaviour following I/R, calculated across all experimental preparations in Aged (burgundy) and Aged pre-treated with the TRPV4 antagonist HC067047 (Aged HC, 1 µmol/L). *P < 0.001 Aged vs. Aged HC, two-tailed Z test of two population proportions (right). Distance scale bars in B, D = 30 µm.

Figure 4

Diastolic Ca²⁺ signals following hypoosmotic stress in cardiomyocytes of aged hearts. (A) Transmitted light image of left ventricular cardiomyocyte isolated from an Aged heart, with cyan rectangle indicating region used for pseudo-line scan images and quantification in panel C. Identical size region in separate cell was used for panel D. (B) Example Fluo-4 fluorescence during diastole (upper) and peak systole (lower) of cardiomyocyte shown in A (300 mOsm/L conditions). (C) Pseudo-line scan images (upper) and ΔF/F₀ fluorescence profiles (lower) of cardiomyocyte of Aged prior to (300 mOsm/L) and following sustained hypoosmotic stress (250 mOsm/L) at t = 3 min, t = 12 min, and t = 15 min. Electrical stimuli indicated by arrowheads; spontaneous (non-paced) Ca²⁺ transients indicated by (*) at t = 12 min. Ca²⁺ waves unresponsive to pacing shown at t = 15 min. Cell death occurred at t = 16 min (not shown). (D) Pseudo-line scan images (upper) and ΔF/F₀ fluorescence profiles (lower) of cardiomyocyte of Aged pre-treated with the TRPV4 antagonist HC067047 (Aged HC, 1 µmol/L), prior to (300 mOsm/L) and following sustained hypoosmotic stress (250 mOsm/L) at t = 3 min, t = 15 min, and t = 30 min. Protocol concluded at 30 min with no cell death in Aged HC. Note modest reduction in ΔF/F₀ at t = 3 min in both Aged (C) and Aged HC (D) in response to changes in osmotic conditions. (E) Summary data of peak Ca²⁺ transient amplitude during 30 min of hypoosmotic stress protocol in Aged (burgundy) and Aged HC (grey). (F) Time to spontaneous diastolic Ca²⁺ events in Aged (burgundy) and Aged HC (grey). (G) Ratio of spontaneous diastolic Ca²⁺ events per electrical stimulation with time in Aged (burgundy) and Aged HC (grey). n = 7 cells from N = 7 animals per group. *P = 0.014 Aged vs. Aged HC, paired two-tailed t test (E). *P = 0.023 Aged vs. Aged HC, One-tailed Wilcoxon matched pairs signed rank test (F). Distance scale bars in A–D = 10 µm.

Figure 5

TRPV4 contributes to arrhythmia following ischaemia–reperfusion in aged hearts. (A) Example left ventricular APs (upper panel), left-atrial electrocardiogram lead (middle panel), and left ventricular electrocardiogram lead (lower panel) of an Aged heart following I/R. Ventricular premature beat highlighted in pink. Example trace taken 19 min into reperfusion phase. (B) Example combined left-atrial/left ventricular electrocardiogram lead (upper panel) and left ventricular electrocardiogram lead (lower panel) of a separate Aged heart following I/R. Ventricular tachycardia highlighted in red. Example trace taken 11 min into reperfusion phase. (C) Summary data demonstrating percentage of total sampled hearts (5/5 Aged vs. 2/5 Aged HC) exhibiting ventricular arrhythmia or ventricular premature beats following I/R. (D) Pie charts indicating percentage of total minutes with at least one ventricular arrhythmic event in Aged (left) and Aged HC (right). (E) Time to arrhythmia in hearts of Aged (burgundy, N = 5) and Aged pre-treated with the TRPV4 antagonist HC067047 (Aged HC, 1 µmol/L, grey, N = 5, left). Arrhythmia score for Aged (burgundy) and Aged HC (grey) shown in early (0–15 min, middle, N = 5 per group) and later (15–30 min, right, N = 4 Aged and N = 5 Aged HC) reperfusion. In 15–30 min time range one Aged heart exhibited AV block and was excluded from Arrhythmia Score analysis. *P = 0.038 Aged vs. Aged HC, two-tailed Z test of two population proportions (C). *P = 0.003 Aged vs. Aged HC, one-tailed unpaired t test (E, left) *P = 0.02 Aged vs. Aged HC, one-tailed Mann–Whitney test (E, middle).