Defects in T-tubular electrical activity underlie local alterations of calcium release in heart failure

Abstract

Action potentials (APs), via the transverse axial tubular system (TATS), synchronously trigger uniform Ca(2+) release throughout the cardiomyocyte. In heart failure (HF), TATS structural remodeling occurs, leading to asynchronous Ca(2+) release across the myocyte and contributing to contractile dysfunction. In cardiomyocytes from failing rat hearts, we previously documented the presence of TATS elements which failed to propagate AP and displayed spontaneous electrical activity; the consequence for Ca(2+) release remained, however, unsolved. Here, we develop an imaging method to simultaneously assess TATS electrical activity and local Ca(2+) release. In HF cardiomyocytes, sites where T-tubules fail to conduct AP show a slower and reduced local Ca(2+) transient compared with regions with electrically coupled elements. It is concluded that TATS electrical remodeling is a major determinant of altered kinetics, amplitude, and homogeneity of Ca(2+) release in HF. Moreover, spontaneous depolarization events occurring in failing T-tubules can trigger local Ca(2+) release, resulting in Ca(2+) sparks. The occurrence of tubule-driven depolarizations and Ca(2+) sparks may contribute to the arrhythmic burden in heart failure.

Keywords: calcium imaging; cardiac disease; nonlinear microscopy; voltage imaging.

Fig. 1.

Simultaneous multisite voltage and Ca²⁺ recording. (A) Scheme of the random access multiphoton (RAMP) microscope. It consists of a 1064-nm fiber laser, an acousto-optic modulator (AOM) for angular-spreading precompensation, and two orthogonally mounted acousto-optic deflectors (AODs) (AOD-x and AOD-y) for laser scanning. The fluorescence signal is collected in forward and backward directions using four independent photomultipliers (PMTs), two for the voltage and two for the calcium signals. (Inset) The emission spectra of the Ca²⁺ probe (dark gray) and VSD (light gray) together with the band-pass filter used for each channel. (B) Two-photon fluorescence (TPF) image of a stained rat ventricular myocyte: sarcolemma in magenta (di-4-ANE(F)PTEA) and [Ca²⁺]_i in green (GFP-certified Fluoforte). (Scale bar, 5 μm.) (C) Normalized fluorescence traces (ΔF/F₀) simultaneously recorded from the scanned sites indicated in white in B: surface sarcolemma (SS) and five T-tubules (TTi). AP is elicited at 200 ms (black arrowheads). Membrane voltage (magenta) and [Ca²⁺]_i (green).

Fig. 2.

Stochastic nature of Ca²⁺ release. (A) Ten subsequent [Ca²⁺]_i fluorescence traces (ΔF/F₀) close to SS and close to a TT of a control (CTRL) and an isoproterenol-treated (ISO) rat cardiomyocyte. Isopreterenol is used at 10⁻⁷M. AP is elicited at 200 ms (black arrowhead). (B) Graphs showing mean values for Ca²⁺ transient time-to-peak (TTP) and 50% of Ca²⁺ decay (CaT₅₀). (C) Graph showing the variability (coefficient of variation, CV) of Ca²⁺ release amplitude (AMP), TTP, and CaT₅₀ in the same probed sites at subsequent stimulated events. Each bar represents the mean ± SE. Data from 27 SS, 124 TTs (27 CTRL cells) and 11 SS, 48 TT (11 ISO cells). Asterisks indicate significant differences (Student t test, *P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 3.

Delay of Ca²⁺ release in AP-failing TT of acute detubulated cells. (A) TPF image of a stained rat ventricular myocyte after formamide-induced osmotic shock; membrane in magenta and [Ca²⁺]_i in green. (Scale bar, 5 μm.) (B) Average of 10 subsequent fluorescence traces (ΔF/F₀) of voltage (magenta) and [Ca²⁺]_i (green) recorded at the three different sites indicated in A: SS and two TTi. APs are elicited at 200 ms (black arrowheads). (C) Superposition of the Ca²⁺ traces nearby SS and TT₁ (Top) and of SS and TT₂ (Middle). (Bottom) A close-up of TT₂ and SS superposition (interval indicated by dashed line in the middle trace). (D) Graphs showing mean values for Ca²⁺ release TTP and CaT₅₀, discriminating electrically coupled (AP+) and uncoupled (AP−) tubules. Ochre lines represent the Ca²⁺ kinetics features measured nearby TT of CTRL: mean (solid) ± SE (dashed). Asterisks indicate significant differences (Student t test, *P < 0.05, **P < 0.01, ***P < 0.001). Ochre asterisks refer to the comparison with CTRL values. Data from 8 SS, 14 AP+ TTs, and 41 AP− TTs (nine cells).

Fig. 4.

Ca²⁺ release in AP-failing TT of HF. (A) TPF image of a stained rat ventricular myocyte isolated from a failing heart: membrane in magenta and [Ca²⁺]_i in green. (Scale bar, 5 μm.) (B) Average of 10 subsequent fluorescence traces (ΔF/F₀) from the scanned lines indicated in A, SS and TTi. APs are elicited at 200 ms (black arrowheads). Membrane voltage in magenta and [Ca²⁺]_i in green. The gray dashed line indicates the Ca²⁺ release time-to-peak measured nearby SS, shown for comparison. (C) Graphs showing Ca²⁺ release TTP and CaT₅₀ nearby SS and TT. The failing TTs (AP−) have been distinguished from the electrically responsive ones (AP+). Ochre lines represent the Ca²⁺ kinetics features measured nearby TT of CTRL: mean (solid) ± SE (dashed). Asterisks indicate significant differences (Student t test, *P < 0.05, **P < 0.01, ***P < 0.001). Ochre asterisks refer to the comparison with CTRL values. Data from 59 SS, 364 AP+ TTs, and 23 AP− TTs (59 HF cells).

Fig. 5.

Ca²⁺ sparks in failing tubules. (A) Representative fluorescence traces (ΔF/F₀) from two CTRL rat cardiomyocytes. The blue arrows highlight the presence of spontaneous Ca²⁺ sparks. AP is elicited at 200 ms (black arrowheads). Membrane voltage (magenta) and [Ca²⁺]_i (green). (B) The trace shows the corresponding membrane potential value of 47 aligned and averaged CTRL Ca²⁺ sparks. The blue arrow shows the alignment point. (C) Graph showing the frequency of Ca²⁺ sparks (νs) occurring in AP+ and AP− tubules of acutely detubulated and HF cells. Each bar represents the mean ± SE. Ochre lines represent the Ca²⁺ spark frequency occurring nearby TTs of CTRL: mean (solid) ± SE (dashed). Asterisks indicate significant differences (Student t test, *P < 0.05, ***P < 0.001). Ochre asterisks refer to the comparison with CTRL values.

Fig. 6.

Voltage-associated Ca²⁺ sparks (V-sparks). (A) Fluorescence traces (ΔF/F₀) from two failing TTs from HF cardiomyocytes and two failing TTs from isoproterenol-treated HF cells (HF+ISO) displaying spontaneous electrical activity. Electrical trigger at 200 ms (black arrowheads). Membrane voltage in magenta and [Ca²⁺]_i in green. (B) Graph showing the frequency of spontaneous depolarization events (νSD) in AP+ and AP− tubules of HF and HF+ISO cells. Each bar represents the mean ± SE. (C) Fluorescence traces (ΔF/F₀) of two failing tubules, one from a HF and one from a HF+ISO displaying voltage-associated Ca²⁺ sparks. (Bottom) Close-ups of the correspondent graphs above (dashed lines interval). Voltage and Ca²⁺ traces have been differently magnified (y axis) to better compare the occurrence timing of the spontaneous events. (D) Graph showing the percentage of spontaneous depolarization events that are associated with a correspondent local Ca²⁺ spark (V-sparks). Each bar represents the mean ± SE.