Background

The cardiomyocyte transverse-tubular system (t-system) couples voltage-gated Ca²⁺ channels in the cell membrane to ryanodine receptors in the sarcoplasmic reticulum, creating the structural precondition for efficient excitation-contraction coupling in the heart. It is established that loss and remodeling of the t-system in heart failure (HF) causes spatiotemporal heterogeneity of intracellular Ca²⁺ release, leading to reduced contractility. However, because t-tubules possess a high density of the Na⁺Ca²⁺ exchanger (NCX), the t-system may also be crucial for cellular Ca²⁺removal and cardiac relaxation. Therefore, we explored contractile kinetics in human myocardial slices and related them to t-system measures. Further, we investigated how Ca²⁺ removal depends on t-tubule distance in human cardiomyocytes.

Methods

Human left-ventricular tissue slices were cultured in vitro under continuous electrical stimulation. Contraction cycles were monitored, analyzed and correlated with intracellular t-tubule distance (DTT) obtained by confocal microscopy. Ca²⁺ transients of isolated human myocytes were recorded by fast line scanning (500fps), after Fluo-4 loading. Membrane staining and confocal imaging served to calculate three-dimensional (3D) DTT of each scanned pixel by registering the scanned line into a 3D image stack of the cell. For local Ca²⁺ kinetics, transients were fitted pixel-wise with a bi-sigmoidal model, allowing to fit not only the Ca²⁺ release, but also the decay with one continuous function. Upstroke and decay parameters were then correlated with the respective DTT of each pixel. Synchrony of Ca²⁺ decay was assessed by the standard deviation of the time when 50% of baseline Ca²⁺ was reached

Results

We found that in myocardial slices from 9 failing human hearts total contraction duration and time-to-relaxation correlated with DTT (R²=0.39 p<0.05 and R²=0.5 p<0.05, respectively), suggesting that an intact t-system is important for myocardial relaxation. This led to the question how cellular Ca²⁺ cycling is affected by DTT. Isolated cardiomyocytes with DTT>1µm, i.e. severe t-system loss, showed significantly slower (859±39ms, n=25) and a less synchronous Ca²⁺ decay (288±19ms, n=26 cells) than cells with DTT<1µm, i.e. higher t-system density (660±53ms, n=49, p<0.01; 212±25ms, n=55 cells, p<0.05, respectively). In accordance with this finding, Ca²⁺ transient duration was increased in cells with high DTT (1.42±0.13s vs 1.75±0.08s, n=20/27 cells, p<0.05). Statistical analysis revealed an increase in Ca²⁺ decay time with increasing DTT of 40±18ms (p<0.001), i.e. Ca²⁺ removal was slower in t-system distant areas. We additionally tested if SERCA inhibition by thapsigargin affects this relationship on the cellular level. Thapsigargin nearly doubled the dependence of Ca²⁺ decay time on t-tubule distance from 45ms/µm to 84ms/µm (p<0.001). 

Conclusion

Reduced t-system density is associated with slowed relaxation in failing human myocardium. On the cellular level, this effect seems to result from a slowed rate of Ca²⁺ removal in regions far from the t-system, which is aggravated when SERCA activity is blocked. We suggest that t-system loss in HF not only impairs cardiac contractility but also affects myocardial relaxation, particularly when SERCA activity is low.