Sheet-Like Remodeling of the Transverse Tubular System in Human Heart Failure Impairs Excitation-Contraction Coupling and Functional Recovery by Mechanical Unloading

Abstract

Background: Cardiac recovery in response to mechanical unloading by left ventricular assist devices (LVADs) has been demonstrated in subgroups of patients with chronic heart failure (HF). Hallmarks of HF are depletion and disorganization of the transverse tubular system (t-system) in cardiomyocytes. Here, we investigated remodeling of the t-system in human end-stage HF and its role in cardiac recovery.

Methods: Left ventricular biopsies were obtained from 5 donors and 26 patients with chronic HF undergoing implantation of LVADs. Three-dimensional confocal microscopy and computational image analysis were applied to assess t-system structure, density, and distance of ryanodine receptor clusters to the sarcolemma, including the t-system. Recovery of cardiac function in response to mechanical unloading was assessed by echocardiography during turndown of the LVAD.

Results: The majority of HF myocytes showed remarkable t-system remodeling, particularly sheet-like invaginations of the sarcolemma. Circularity of t-system components was decreased in HF versus controls (0.37±0.01 versus 0.46±0.02; P<0.01), and the volume/length ratio was increased in HF (0.36±0.01 versus 0.25±0.02 µm²; P<0.0001). T-system density was reduced in HF, leading to increased ryanodine receptor-sarcolemma distances (0.96±0.05 versus 0.64±0.1 µm; P<0.01). Low ryanodine receptor-sarcolemma distances at the time of LVAD implantation predicted high post-LVAD left ventricular ejection fractions (P<0.01) and ejection fraction increases during unloading (P<0.01). Ejection fraction in patients with pre-LVAD ryanodine receptor-sarcolemma distances >1 µm did not improve after mechanical unloading. In addition, calcium transients were recorded in field-stimulated isolated human cardiomyocytes and analyzed with respect to local t-system density. Calcium release in HF myocytes was restricted to regions proximal to the sarcolemma. Local calcium upstroke was delayed (23.9±4.9 versus 10.3±1.7 milliseconds; P<0.05) and more asynchronous (18.1±1.5 versus 8.9±2.2 milliseconds; P<0.01) in HF cells with low t-system density versus cells with high t-system density.

Conclusions: The t-system in end-stage human HF presents a characteristic novel phenotype consisting of sheet-like invaginations of the sarcolemma. Our results suggest that the remodeled t-system impairs excitation-contraction coupling and functional recovery during chronic LVAD unloading. An intact t-system at the time of LVAD implantation may constitute a precondition and predictor for functional cardiac recovery after mechanical unloading.

Keywords: excitation contraction coupling; heart failure; myocytes, cardiac; recovery of function; ryanodine receptor calcium release channels.

Figure 1. Two-dimensional tile scan of WGA-labeled LV tissue slices obtained from control and HF patient at time of LVAD implantation

Images were acquired with a confocal microscope equipped with a 40× oil immersion lens using a pixel size of 0.2×0.2μm. (A) Tile scan from donor tissue. (B) Magnified view of the boxed region in (A) showing myocytes with dense t-system. (C) Tile scan from HF tissue. (D,E) Magnified views of the boxed region in (C) with myocytes with a sparse and irregular t-system. The remodeled t-system exhibited longitudinal components in the majority of cells. Some examples were marked with arrows. Scale bar in A is 500μm and also applies to C. Scale bar in B is 50μm and also applies to D and E.

Figure 2. Three-dimensional imaging and reconstruction of t-system from control and HF tissue

(A) Confocal microscopic image from control tissue labeled with WGA (red) for extracellular matrix and sarcolemma. A segmented cardiomyocyte is highlighted in green. (B) Magnified view of the boxed regions in (A) showing myocyte with a dense t-system. (C) 3D reconstruction of outer sarcolemma (gray) and t-system (blue) of a section extracted from the cell highlighted in (A). (D) T-tubule highlighted in (C) shown in longitudinal and transverse view. (E) Image from HF tissue labeled as in (A). A segmented cardiomyocyte is highlighted in green. (F) Magnified view of the boxed region in (E) reveals sparse t-system with sheet-like remodeling. Black arrows point to the same sheet-like component of the t-system. (G) 3D reconstruction of outer sarcolemma (gray) and t-system (blue) of a section from the cell highlighted in (E). (H) Remodeled t-system component highlighted in (G) shown in longitudinal and transverse view. Scale bar in A: 40μm. Applies to E. Scale bar in B: 5μm. Applies to F. Scale bar in C: 10μm. Applies to G. Scale bar in D: 2μm. Applies to H.

Figure 3. Three-dimensional analyses of t-tubule geometry in control and HF tissue and imaging of isolated cardiomyocytes

Using an extracellular marker confirm remodeling of t-tubules. (A) Cross-sectional circularity of t-tubules, calculated from the two minor eigenvalues |λ₃/λ₂|, was lower in HF (n=26) than in control (n=5). (B) Volume-length ratio (V/l) of t-tubules was increased in HF vs control. (C) Mean intracellular sarcolemma distance (Δ_SL) was higher in HF cells than in control cells. (D) Δ_SL increased with V/l (p<0.01 vs constant model). (E) XY, ZY and XZ cross-sections through three-dimensional image of living isolated cardiomyocyte from HF patient. Dashed blue lines indicate cross-sections. The cell was bathed in 5mg/ml dextran-FITC conjugate (10kDa) as an extracellular marker. Extracellular fluid is shown in white, intracellular space in black. T-sheets were also present in isolated cells (example marked with blue arrow) and exhibited fluorescence, indicating that t-sheet cavities are connected to the extracellular space. Scale bar is 20μm. **p<0.01, ****p<0.0001

Figure 4. Analyses of RyR-sarcolemma distances in tissue from donors and pre-LVAD patients

Confocal microscopic images of (A) control and (C) HF LV tissue labeled with WGA (red) for extracellular matrix and sarcolemma, and for RyRs (green). (B and D) Magnified view of the boxed regions in (A) and (C), respectively. (E) Fraction of RyR fluorescence intensity (I_RyR) found at defined sarcolemma distances (Δ_SL) in control (black, n=5) and HF (white, n=26). (F) RyR-sarcolemma distance (Δ_RyR-SL) in control and HF. Scale bar in A: 40μm. Applies to C. Scale bar in B: 5μm. Applies to D. *p<0.05, **p<0.01

Figure 5. Statistical analyses of clinical and imaging data

Linear regression models of pre-LVAD RyR-sarcolemma distance (Δ_RyR-SL) versus (A) pre-LVAD EF (EF_pre), (B) post-LVAD EF (EF_post), and (C) EF change during unloading (ΔEF=EF_post−EF_pre). Significance of F-statistics against constant model is indicated by p and correlation coefficient by R. Dotted lines indicate 95% confidence intervals. (D) pre-LVAD EF, (E) post-LVAD EF, (F) EF change, (G) HF duration (t_HF), and (H) sarcolemma distance (Δ_SL) in samples from patients grouped by low (<1μm, black, n=16) and high (>1μm, white, n=10) RyR-sarcolemma distance. **p<0.01 (<1μm vs >1μm), † p<0.001 (EF_post vs EF_pre)

Figure 6. Imaging and analyses of calcium transients in HF cardiomyocytes

(A) Confocal microscopic image of Di-8-ANEPPS labeled isolated myoycte with sheet-like remodeled t-system. (B) Fluo-4 images of cell at time 0, 36 and 98ms after stimulation. (C) Map of onset times of Fluo-4 signal (t_onset). (D) Map of maximal upstroke velocities in Fluo-4 signal (du/dt_max). (E) Mean±SD of t_onset and (F) du/dt_max versus sarcolemma distance (Δ_SL). Statistical analysis were performed on 15 HF myocytes. (G) Mean±SD of t_onset and (H) du/dt_max versus Δ_SL. (I) t_onset and (J) its standard deviation (σt_onset) in cells grouped by low (<1μm, black) and high (>1μm, white) Δ_SL. Scale bar in A: 10μm. Applies to B–D. *p<0.05, **p<0.01

Figure 7. Proposed role of the t-system in functional cardiac recovery in chronic human HF

In some failing hearts, normal t-tubules remodel to t-sheets, t-system components that are significantly widened in the myocyte long axis direction, but still run transversely. Concomitantly, t-system density decreases, which causes a higher fraction of non-junctional ryanodine receptor (RyR) clusters. As a result, triggering of calcium release from the sarcoplasmic reticulum (SR) becomes less efficient. Mechanical unloading by LVAD allows recovery of metabolism, beta-adrenergic response and calcium transporters, but not of t-system structure. Thus, excitation-contraction (EC) coupling can only recover in failing hearts with a preserved t-system, leading to improved contractility and left-ventricular ejection fraction (EF) following unloading, but not in failing hearts with t-sheets and reduced t-system density.