Ca2+ transient features are improved after mechanical unloading
Heart failure cells showed a depressed Ca2+ transient amplitude, which improved in HF-UN. An important determinant of the Ca2+ transient amplitude is SR Ca2+ content, which was depressed in HF and increased towards sham values in HF-UN (ratio units: sham, 0.212 ± 0.07, n = 53; HF, 0.162 ± 0.04, n = 20; HF-UN, 0.238 ± 0.05, n = 47; sham vs. HF, P = 0.01, HF vs. HF-UN, P = 0.001; sham and HF-UN were not statistically different). HF cells showed a prolonged time to peak of the Ca2+ transient as well as time to 50% and 90% decline in the Ca2+ transient. These features were also normalized by HF-UN (Figure ). The variance of the time-to-peak of the Ca2+ transient measured at each pixel is taken as an index of CICR dyssynchrony and was increased in HF cells, but recovered in HF-UN (Figure ). This suggests that the stimulus activates Ca2+ release throughout the cell more uniformly in HF-UN. There are a number of possible causes for this normalization of CICR including normalized L-type Ca2+ channel activity or normalized RyR function. Another possibility is that coupling between these trigger and release sites, which partly sets the gain of the positive feedback component of CICR, is altered.
Raised Ca2+ spark frequency is reduced by mechanical unloading
The spontaneous opening of RyR clusters can be characterized by measuring Ca2+ sparks. Ca2+ spark frequency was increased in HF cells compared with sham cells, but normalized after HF-UN (Figure ). HF cells had a significantly higher Ca2+ spark peak amplitude compared with sham myocytes, but unloading did not decrease Ca2+ spark peak amplitude. Ca2+ spark width and duration were increased by HF and mechanical unloading (Figure ).
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 (more ...)
Mechanical unloading causes a regression of cellular hypertrophy
We assessed the volume of single cardiomyocytes using three-dimensional reconstruction of di-8-ANNEPPS images. Mechanical unloading induced a regression of hypertrophy, and average cell volume was smaller than for sham cells (Figure ).
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 (more ...)
Depressed L-type Ca2+ channel activity is rescued by mechanical unloading
current activity was depressed in HF and was normalized in HF-UN (Figure
). Cell capacitance, an index of cell surface area, showed a normalization to sham levels in HF-UN (Figure
). The rate of activation was unaffected (data not shown), but the rate of fast, Ca2+
-dependent inactivation was faster in HF and was normalized in HF-UN (Figure
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 (more ...)
Mechanical unloading restores normal transverse tubule structure
Heart failure reduced the t-tubule density significantly compared with sham, and this was recovered by HF-UN (Figure ). The deterioration in the regularity of the t-tubule system in HF, as measured by the power of the dominant frequency of the Fourier transform, was also normalized by HF-UN (Figure ).
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 (more ...)
Transverse tubule improvements after mechanical unloading are accompanied by improved cell surface structure
In sham cells, clearly defined z-grooves were present which contain the t-tubule openings. This was characterized by a high z-groove index. The cell surface was flattened and distorted in HF, with a reduction in the z-groove index. These features recovered after HF-UN (Figure ).
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 (more ...)
Mechanical unloading restores normal transverse tubule microarchitecture
Transverse tubule microarchitecture was assessed using transmission electron microscopy of single cardiomyocytes. The t-tubule lumens were reduced in density and dilated in HF. HF-UN normalized these parameters towards sham levels (Figure ).
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 (more ...)
Mechanical unloading restores dihydropyridine receptor–ryanodine receptor coupling
Because the repair of the t-tubule system was associated with functional improvements to the CICR process, we assessed whether structural restoration of the DHPR–RyR relationship might account for the improved cellular Ca2+ handling. In HF, the degree of co-localization of DHPRs and RyRs was reduced, but partially recovered in HF-UN (Figure ).
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 (more ...)
Junctophilin-2 is not directly responsible for improved dyadic coupling
Junctophilin-2 has been proposed as a regulator of the t-tubule system which is responsible for coupling t-tubule and SR membranes. Its expression is reduced with the progression from hypertrophy to HF,8
possibly due to increasing overload. To test whether the improvements in t-tubule structure were due to changes in JP-2 expression, we performed western blotting (Supplementary material online, Figure S1
). JP-2 expression was significantly reduced in HF, but not recovered by HF-UN.