Subcellular Heterogeneity of Ryanodine Receptor Properties in Ventricular Myocytes with Low T-Tubule Density

Liesbeth Biesmans,Virginie Bito,Frank R Heinzel,Godfrey L Smith,Karin R Sipido,Niall Macquaide,Marcello Rota

doi:10.1371/journal.pone.0025100

Liesbeth Biesmans, Virginie Bito + Show 5 more

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https://doi.org/10.1371/journal.pone.0025100

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Abstract

RationaleIn ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI).ObjectiveTo characterize RyR functional properties in relation to TT proximity, at baseline and after MI.MethodsMyocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca2+ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca2+ release, F>F50 within 20 ms) or their absence (delayed areas). Spontaneous Ca2+ release events during diastole, Ca2+ sparks, reflecting RyR activity and properties, were subsequently assigned to either category.ResultsIn CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na+/Ca2+ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca2+ influx via Ca2+ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca2+ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca2+ removal by NCX at the membrane was significantly lower in MI.ConclusionTT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca2+ loss and raise SR Ca2+ content, but may promote Ca2+ waves.

Highlights

In ventricular cardiac myocytes, Ca2+ influx through voltage-gated L-type Ca2+ channels (LTCC) activates Ca2+ release from ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR)
We build on our earlier data where we identified areas without TTs within pig ventricular myocytes based on the local Ca2+ transient properties during normally triggered SR Ca2+ release [12,18]
Since Na+/ Ca2+ exchanger (NCX) is known to be highly expressed in TTs [2,21], we investigated whether NCX affects spark duration preferentially in early release areas

Summary

Introduction

Ca2+ influx through voltage-gated L-type Ca2+ channels (LTCC) activates Ca2+ release from ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR). This Ca2+induced Ca2+ release occurs in local release units or couplons, i.e. clusters of LTCC facing clusters of RyR, which are preferentially located in the T-tubules (TT). TTs are poorly developed or absent in neonatal myocytes and in cells from the specialized conduction system such as Purkinje cells, as well as in atrial cells These cells are small and show a radial spread of Ca2+ from the external sarcolemma to the center mediated by a combination of Ca2+ release and diffusion [7,8]

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