Hoang-Trong M. Tuan

STED Live Cell Super-Resolution Imaging Shows Proliferative Remodeling of T-Tubule Membrane Structures After Myocardial Infarction

Rationale: Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca2+ release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). Objectives: Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. Methods and Results: Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca2+ release and action potential prolongation. Conclusions: This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca2+ release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing.

Ryanodine receptor cluster fragmentation and redistribution in persistent atrial fibrillation enhance calcium release

Aims In atrial fibrillation (AF), abnormalities in Ca2+ release contribute to arrhythmia generation and contractile dysfunction. We explore whether ryanodine receptor (RyR) cluster ultrastructure is altered and is associated with functional abnormalities in AF. Methods and results Using high-resolution confocal microscopy (STED), we examined RyR cluster morphology in fixed atrial myocytes from sheep with persistent AF (N = 6) and control (Ctrl; N = 6) animals. RyR clusters on average contained 15 contiguous RyRs; this did not differ between AF and Ctrl. However, the distance between clusters was significantly reduced in AF (288 ± 12 vs. 376 ± 17 nm). When RyR clusters were grouped into Ca2+ release units (CRUs), i.e. clusters separated by <150 nm, CRUs in AF had more clusters (3.43 ± 0.10 vs. 2.95 ± 0.02 in Ctrl), which were more dispersed. Furthermore, in AF cells, more RyR clusters were found between Z lines. In parallel experiments, Ca2+ sparks were monitored in live permeabilized myocytes. In AF, myocytes had >50% higher spark frequency with increased spark time to peak (TTP) and duration, and a higher incidence of macrosparks. A computational model of the CRU was used to simulate the morphological alterations observed in AF cells. Increasing cluster fragmentation to the level observed in AF cells caused the observed changes, i.e. higher spark frequency, increased TTP and duration; RyR clusters dispersed between Z-lines increased the occurrence of macrosparks. Conclusion In persistent AF, ultrastructural reorganization of RyR clusters within CRUs is associated with overactive Ca2+ release, increasing the likelihood of propagating Ca2+ release.

Stochastic simulation of cardiac ventricular myocyte calcium dynamics and waves

A three dimensional model of calcium dynamics in the rat ventricular myocyte was developed to study the mechanism of calcium homeostasis and pathological calcium dynamics during calcium overload. The model contains 20,000 calcium release units (CRUs) each containing 49 ryanodine receptors. The model simulates calcium sparks with a realistic spontaneous calcium spark rate. It suggests that in addition to the calcium spark-based leak, there is an invisible calcium leak caused by the stochastic opening of a small number of ryanodine receptors in each CRU without triggering a calcium spark. The model also explores the mechanism of calcium wave propagation between release sites under the conditions of calcium overload.

Ca2+ Leak and Ca2+ Sparks in Mammalian Heart: Insights from a Computational Model

Calcium (Ca2+) signaling in muscle, neuronal, and non-excitable cells has benefited significantly from advances in biological tools and imaging technology, however, the molecular interactions of nanoscopic molecules, structures and compartments has been challenging to study under physiological conditions. Here, we exploit novel computational modeling techniques to examine real-time molecular and cellular physiology in cardiac ventricular myocytes. The model focuses on local and cell-wide Ca2+ signaling phenomena related to calcium induced calcium release from intracellular calcium channels, ryanodine receptors (RyR2s), located on the sarcoplasmic reticulum (SR) membrane. This work is informed by the latest molecular investigations and recent characterizations of channels, transporters, and buffers located in mammalian heart. We have created a detailed, whole-cell model of Ca2+ signaling using a realistic number of calcium release units (CRU) each containing a cluster of stochastically gating RyR2s. During systole the opening of these RyR2s is triggered by Ca2+ entry via voltage gated L-type Ca2+ channels. The synchronized opening of the RyR2 cluster leads to localized elevations of [Ca2+]i known as Ca2+ sparks. During diastole Ca2+ sparks are still observed and are attributed to the finite opening rate of the RyR2. RyR2s are also believed to display unsynchronized or non-spark openings where only a few channels in the CRU open without triggering the remainder of the RyR2 cluster. This non-spark Ca2+ release would be below current experimental detection thresholds and therefore “invisible.” These spark and non-spark openings of RyR2s constitute a molecular basis for Ca2+ leak from the SR. The computational model suggests that a significant fraction of SR Ca2+ leak is due to RyR2s openings that fail to trigger a “visible” Ca2+ spark. Additionally, the fraction of non-spark or “invisible” SR Ca2+ leak increases as SR Ca2+ content declines.