Loss of T-tubules and other changes to surface topography in ventricular myocytes from failing human and rat heart

Abstract

T-tubular invaginations of the sarcolemma of ventricular cardiomyocytes contain junctional structures functionally coupling L-type calcium channels to the sarcoplasmic reticulum calcium-release channels (the ryanodine receptors), and therefore their configuration controls the gain of calcium-induced calcium release (CICR). Studies primarily in rodent myocardium have shown the importance of T-tubular structures for calcium transient kinetics and have linked T-tubule disruption to delayed CICR. However, there is disagreement as to the nature of T-tubule changes in human heart failure. We studied isolated ventricular myocytes from patients with ischemic heart disease, idiopathic dilated cardiomyopathy, and hypertrophic obstructive cardiomyopathy and determined T-tubule structure with either the fluorescent membrane dye di-8-ANNEPs or the scanning ion conductance microscope (SICM). The SICM uses a scanning pipette to produce a topographic representation of the surface of the live cell by a non-optical method. We have also compared ventricular myocytes from a rat model of chronic heart failure after myocardial infarction. T-tubule loss, shown by both ANNEPs staining and SICM imaging, was pronounced in human myocytes from all etiologies of disease. SICM imaging showed additional changes in surface structure, with flattening and loss of Z-groove definition common to all etiologies. Rat myocytes from the chronic heart failure model also showed both T-tubule and Z-groove loss, as well as increased spark frequency and greater spark amplitude. This study confirms the loss of T-tubules as part of the phenotypic change in the failing human myocyte, but it also shows that this is part of a wider spectrum of alterations in surface morphology.

Fig. 1.

SICM images from the surface of cardiomyocytes isolated from nonfailing (A) and failing (B) human hearts. The black dotted line represents the linear selection presented as a 1-dimensional surface contour map from nonfailing (C) and failing (E) human cardiomyocytes. Confocal images after staining with di-8-ANNEPPS in nonfailing (D) and failing cardiomyocytes (F). T-tubule (G) and Z-groove (H) ratios in cardiomyocytes isolated from patients with DCM, HF secondary to IHD, or HOCM. NF, nonfailing. (I) Prolonged TTP and relaxation times (R50 and R90) in human failing cardiomyocytes (solid bars, n = 12) compared with nonfailing human cardiomyocytes (open bars, n = 6). **, P < 0.01 vs. nonfailing.

Fig. 2.

The rat chronic post–myocardial infarction (MI) HF model. (A) Midventricular 10-μm section from a sham control rat heart (Left) and a chronically infarcted rat heart (Right) after staining with Masson's trichrome. (Scale bar, 2 mm.) (B) Representative in vivo PV loops during transient inferior vena caval occlusion from an HF rat and a Sham control. ESPVR (red broken lines) and EDPVR (black broken lines) relationships are presented. (C) Representative in vivo steady-state PV loops demonstrating increased ventricular volumes and elevated end-diastolic pressure in HF rats (black arrow) compared with Sham controls (red arrow). (D) Steady-state PV data demonstrating decreased LVEF, increased LVEDP, and reduced peak velocities of pressure change (dPdt) during isovolumic contraction (Peak + dPdt) and isovolumic relaxation (Peak − dPdt) in rats with HF. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig. 3.

(A) Representative tracings from stimulated (0.5 Hz) isolated rat cardiomyocytes demonstrating reduced amplitude and prolonged relaxation in cardiomyocytes from failing hearts. Mean contraction amplitude in isolated cardiomyocytes from sham-ligated (white bars; n = 11 cells) and failing (black bars; n = 14 cells) rat hearts. (B) Cytoplasmic Ca²⁺ transient data with TTP, R50, and R90 in isolated cardiomyocytes from sham-ligated (white bars) and failing (black bars) rat hearts. (C) Representative examples of confocal line scan images demonstrating spontaneous Ca²⁺ sparks from failing (Top) and control (Bottom) cardiomyocytes. Spontaneous Ca²⁺ spark frequency (D) and amplitude (E) in cardiomyocytes from sham-ligated (white bars) and failing (black bars) rat hearts. (F) Images show the onset of 2 sample transients taken from control and HF cells, illustrating synchronous and less-homogeneous release. Regions of delayed Ca²⁺ release have been quantified as detailed in Materials and Methods and averaged from 10 sham control cells and 8 HF cells. *, P < 0.05 vs. HF.

Fig. 4.

SICM images from the surface of cardiomyocytes isolated from sham-ligated (A) and failing (B) rat hearts. Confocal images from a section of the sarcolemmal membrane after staining with di-8-ANNEPPS in control (C) and failing myocytes (D). The T-tubule (E) and Z-groove (F) ratio for sham (open bars; n = 12) and failing myocytes (n = 16) (***, P < 0.001 vs. sham).