Mechanical unloading reverses transverse tubule remodelling and normalizes local Ca(2+)-induced Ca(2+)release in a rodent model of heart failure

Abstract

Aims: Ca(2+)-induced Ca(2+) release (CICR) is critical for contraction in cardiomyocytes. The transverse (t)-tubule system guarantees the proximity of the triggers for Ca(2+) release [L-type Ca(2+) channel, dihydropyridine receptors (DHPRs)] and the sarcoplasmic reticulum Ca(2+) release channels [ryanodine receptors (RyRs)]. Transverse tubule disruption occurs early in heart failure (HF). Clinical studies of left ventricular assist devices in HF indicate that mechanical unloading induces reverse remodelling. We hypothesize that unloading of failing hearts normalizes t-tubule structure and improves CICR.

Methods and results: Heart failure was induced in Lewis rats by left coronary artery ligation for 12 weeks; sham-operated animals were used as controls. Failing hearts were mechanically unloaded for 4 weeks by heterotopic abdominal heart transplantation (HF-UN). HF reduced the t-tubule density measured by di-8-ANEPPS staining in isolated left ventricular myocytes, and this was reversed by unloading. The deterioration in the regularity of the t-tubule system in HF was also reversed in HF-UN. Scanning ion conductance microscopy showed the reappearance of normal surface striations in HF-UN. Electron microscopy revealed recovery of normal t-tubule microarchitecture in HF-UN. L-type Ca(2+) current density, measured using whole-cell patch clamping, was reduced in HF but unaffected by unloading. The variance of the time-to-peak of the Ca(2+) transient, an index of CICR dyssynchrony, was increased in HF and normalized by unloading. The increased Ca(2+) spark frequency observed in HF was reduced in HF-UN. These results could be explained by the recoupling of orphaned RyRs in HF, as indicated by immunofluorescence.

Conclusions: Our data show that mechanical unloading of the failing heart reverses the pathological remodelling of the t-tubule system and improves CICR.

Figure 1

Mechanical unloading (HF-UN) induces a normalization of Ca²⁺ transient parameters. The time-to-peak, decay to 50%, and decay to 90% of the Ca²⁺ transient were prolonged in heart failure (HF). Mechanical unloading normalized these parameters. The amplitude of the Ca²⁺ transient was depressed in HF and normalized by mechanical unloading. The variance of the time-to-peak, an index of Ca²⁺-induced Ca²⁺ release (CICR) synchronicity, was increased in HF and normalized by mechanical unloading. The vertical scale bar indicates 200 ms, and the horizontal scale bar indicates 100 pixels (sham n = 40, HF n = 34, HF-UN n = 29).

Figure 2

Mechanical unloading (HF-UN) normalizes Ca²⁺ spark frequency. Heart failure (HF) cells showed a higher Ca²⁺ spark frequency, peak, width, and duration. Mechanical unloading normalized Ca²⁺ spark frequency and increased width and duration. Mechanical unloading did not influence Ca²⁺ spark peak. The vertical scale bar indicates 280 ms and the horizontal scale bar indicates 100 pixels (sham n = 45, HF n = 34, HF-UN n = 36).

Figure 3

Mechanical unloading (HF-UN) caused a regression of cellular hypertrophy. Mechanical unloading caused a regression of the cellular hypertrophy observed in heart failure (HF), to below sham levels. The cell capacitance, a measure of cell area, was increased in HF and reduced by mechanical unloading. For cell volume, sham n = 39, HF n = 40, HF-UN n = 47; for cell capacitance, sham n = 33, HF n = 26, HF-UN n = 35).

Figure 4

Mechanical unloading (HF-UN) recovers the depressed L-type Ca²⁺ channel density observed in heart failure (HF). Raw traces of L-type Ca²⁺ current are shown. The L-type Ca²⁺ current density was reduced in HF and normalized by mechanical unloading (sham n = 33, HF n = 26, HF-UN n = 35). Fast tau (Ca²⁺-dependent) inactivation is shown in the bottom left.

Figure 5

Mechanical unloading (HF-UN) restores the normal transverse tubule (t-tubule) density and regularity. Single ventricular cardiomyocytes stained with di-8-ANEPPS are shown. Heart failure (HF) resulted in a reduced t-tubule density and a lower power of the dominant peak of the Fourier transform of the t-tubules (an index of t-tubular regularity). Mechanical unloading improved t-tubule density and regularity (sham n = 40, HF n = 32, HF-UN n = 27).

Figure 6

Mechanical unloading (HF-UN) restores normal cell surface architecture. The z-groove index is an index of the regularity of the cell surface. This was reduced in heart failure (HF) and normalized by mechanical unloading (sham n = 14, HF n = 25, HF-UN n = 17).

Figure 7

Mechanical unloading (HF-UN) restores transverse tubular (t-tubular) microarchitecture. Optical sections of transmission electron micrographs of isolated ventricular cardiomyocytes are shown. Heart failure (HF) cells showed fewer total t-tubules per optical section which were more dilated. Mechanical unloading caused a regression of t-tubular dilation, and a restoration of their normal density. Black scale bars indicate 1 µm (80 optical sections were analysed in each animal in each group). M, mitochondria; t, t-tubule.

Figure 8

Mechanical unloading (HF-UN) recouples orphaned ryanodine receptors (RyRs) from dihydropyridine receptors (DHPRs). Examples of RyR (top panel), DHPR (middle panel), and co-localized pixels (CP, lower panel) are shown. In heart failure (HF), DHPR–RyR co-localization is reduced, with a loss of the striated pattern of co-localized pixels. Mechanical unloading significantly improves DHPR–RyR co-localization (sham n = 63, HF n = 49, HF-UN n = 42).