Cardiac Resynchronization Therapy Reduces Subcellular Heterogeneity of Ryanodine Receptors, T-Tubules, and Ca2+ Sparks Produced by Dyssynchronous Heart Failure.

Background: Cardiac resynchronization therapy (CRT) is a major advance for treatment of patients with dyssynchronous heart failure (DHF). However, our understanding of DHF-associated remodeling of subcellular structure and function and their restoration after CRT remains incomplete.

Methods And Results: We investigated subcellular heterogeneity of remodeling of structures and proteins associated with excitation-contraction coupling in cardiomyocytes in DHF and after CRT.

Na+/Ca2+ exchange and Na+/K+-ATPase in the heart.

This paper is the third in a series of reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation-contraction coupling and arrhythmias: Na(+) channel and Na(+) transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on cardiac Na(+)/Ca(2+) exchange (NCX) and Na(+)/K(+)-ATPase (NKA).

Neuronal Na+ channel blockade suppresses arrhythmogenic diastolic Ca2+ release.

Aims: Sudden death resulting from cardiac arrhythmias is the most common consequence of cardiac disease. Certain arrhythmias caused by abnormal impulse formation including catecholaminergic polymorphic ventricular tachycardia (CPVT) are associated with delayed afterdepolarizations resulting from diastolic Ca2+ release (DCR) from the sarcoplasmic reticulum (SR). Despite high response of CPVT to agents directly affecting Ca2+ cycling, the incidence of refractory cases is still significant.

Na/Ca exchange and contraction of the heart.

Sodium-calcium exchange (NCX) is the major calcium (Ca) efflux mechanism of ventricular cardiomyocytes. Consequently the exchanger plays a critical role in the regulation of cellular Ca content and hence contractility. Reductions in Ca efflux by the exchanger, such as those produced by elevated intracellular sodium (Na) in response to cardiac glycosides, raise sarcoplasmic reticulum (SR) Ca stores.

Quantitative analysis of cardiac tissue including fibroblasts using three-dimensional confocal microscopy and image reconstruction: towards a basis for electrophysiological modeling.

Electrophysiological modeling of cardiac tissue is commonly based on functional and structural properties measured in experiments. Our knowledge of these properties is incomplete, in particular their remodeling in disease. Here, we introduce a methodology for quantitative tissue characterization based on fluorescent labeling, 3-D scanning confocal microscopy, image processing and reconstruction of tissue micro-structure at sub-micrometer resolution.

Mechanical modulation of the transverse tubular system of ventricular cardiomyocytes.

In most mammalian cardiomyocytes, the transverse tubular system (t-system) is a major site for electrical signaling and excitation-contraction coupling. The t-system consists of membrane invaginations, which are decorated with various proteins involved in excitation-contraction coupling and mechano-electric feedback. Remodeling of the t-system has been reported for cells in culture and various types of heart disease.

Strain transfer in ventricular cardiomyocytes to their transverse tubular system revealed by scanning confocal microscopy.

The transverse tubular system (t-system) is a major site for signaling in mammalian ventricular cardiomyocytes including electrical signaling and excitation-contraction coupling. It consists of membrane invaginations, which are decorated with various proteins including mechanosensitive ion channels. Here, we investigated mechanical modulation of the t-system.

Na+ currents are required for efficient excitation-contraction coupling in rabbit ventricular myocytes: a possible contribution of neuronal Na+ channels.

Ca2+ transients were activated in rabbit ventricular cells by a sequence of action potential shaped voltage clamps. After activating a series of control transients, Na+ currents (INa) were inactivated with a ramp from -80 to -40 mV (1.5 s) prior to the action potential clamp.

Sodium-calcium exchange is essential for effective triggering of calcium release in mouse heart.

In cardiac myocytes, excitation-contraction coupling depends upon sarcoplasmic reticular Ca2+ release triggered by Ca2+ influx through L-type Ca2+ channels. Although Na+-Ca2+ exchange (NCX) is essential for Ca2+ extrusion, its participation in the trigger process of excitation-contraction coupling is controversial. To investigate the role of NCX in triggering, we examined Ca2+ sparks in ventricular cardiomyocytes isolated from wild-type (WT) and cardiac-specific NCX knockout (KO) mice.

Activation of reverse Na+-Ca2+ exchange by the Na+ current augments the cardiac Ca2+ transient: evidence from NCX knockout mice.

The hypothesis that Na(+) influx during the action potential (AP) activates reverse Na(+)-Ca(2+) exchange (NCX) and subsequent entry of trigger Ca(2+) is controversial. We tested this hypothesis by monitoring intracellular Ca(2+) before and after selective inactivation of I(Na) prior to a simulated action potential in patch-clamped ventricular myocytes isolated from adult wild-type (WT) and NCX knockout (KO) mice. First, we inactivated I(Na) using a ramp prepulse to 45 mV.

Towards computational modeling of excitation-contraction coupling in cardiac myocytes: reconstruction of structures and proteins from confocal imaging.

Computational models of excitation-contraction (EC) coupling in myocytes are valuable tools for studying the signaling cascade that transduces transmembrane voltage into mechanical responses. A key component of these models is the appropriate description of structures involved in EC coupling, such as the sarcolemma and ion channels. This study aims at developing an approach for spatial reconstruction of these structures.

Novel features of the rabbit transverse tubular system revealed by quantitative analysis of three-dimensional reconstructions from confocal images.

With scanning confocal microscopy we obtained three-dimensional (3D) reconstructions of the transverse tubular system (t-system) of rabbit ventricular cells. We accomplished this by labeling the t-system with dextran linked to fluorescein or, alternatively, wheat-germ agglutinin conjugated to an Alexa fluor dye. Image processing and visualization techniques allowed us to reconstruct the t-system in three dimensions.

Sub-micrometer anatomical models of the sarcolemma of cardiac myocytes based on confocal imaging.

We describe an approach to develop anatomical models of cardiac cells. The approach is based on confocal imaging of living ventricular myocytes with submicrometer resolution, digital image processing of three-dimensional stacks with high data volume, and generation of dense triangular surface meshes representing the sarcolemma including the transverse tubular system. The image processing includes methods for deconvolution, filtering and segmentation.

Allosteric activation of Na+-Ca2+ exchange by L-type Ca2+ current augments the trigger flux for SR Ca2+ release in ventricular myocytes.

The possible contribution of Na(+)-Ca(2+) exchange to the triggering of Ca(2+) release from the sarcoplasmic reticulum in ventricular cells remains unresolved. To gain insight into this issue, we measured the "trigger flux" of Ca(2+) crossing the cell membrane in rabbit ventricular myocytes with Ca(2+) release disabled pharmacologically. Under conditions that promote Ca(2+) entry via Na(+)-Ca(2+) exchange, internal [Na(+)] (10 mM), and positive membrane potential, the Ca(2+) trigger flux (measured using a fluorescent Ca(2+) indicator) was much greater than the Ca(2+) flux through the L-type Ca(2+) channel, indicating a significant contribution from Na(+)-Ca(2+) exchange to the trigger flux.

Effect of metabolic inhibition on couplon behavior in rabbit ventricular myocytes.

We investigated the effect of combined inhibition of oxidative and glycolytic metabolism on L-type Ca(2+) channels (LCCs) and Ca(2+) spikes in isolated patch-clamped rabbit ventricular myocytes. Metabolic inhibition (MI) reduced LCC open probability, increased null probability, increased first latency, and decreased open time but left conductance unchanged. These results explain the reduction in macroscopic Ca(2+) current observed during MI.

Variability in couplon size in rabbit ventricular myocytes.

Variation in couplon size is thought to be essential for graded Ca(2+) transients in cardiac myocytes. We examined this variation by investigating spark appearance in rabbit ventricular myocytes at various locations and at potentials from -20 to 0 mV. At 0 mV, sparks appeared at the beginning of the voltage step with a probability of unity.

Metabolic inhibition alters subcellular calcium release patterns in rat ventricular myocytes: implications for defective excitation-contraction coupling during cardiac ischemia and failure.

Metabolic inhibition (MI) contributes to contractile failure during cardiac ischemia and systolic heart failure, in part due to decreased excitation-contraction (E-C) coupling gain. To investigate the underlying mechanism, we studied subcellular Ca2+ release patterns in whole cell patch clamped rat ventricular myocytes using two-dimensional high-speed laser scanning confocal microscopy. In cells loaded with the Ca2+ buffer EGTA (5 mmol/L) and the fluorescent Ca2+-indicator fluo-3 (1 mmol/L), depolarization from -40 to 0 mV elicited a striped pattern of Ca2+ release.

Ca2+ sparks in rabbit ventricular myocytes evoked by action potentials: involvement of clusters of L-type Ca2+ channels.

It is not clear how many L-type Ca2+ channels (LCCs) are required to ensure that a Ca2+ spark is triggered during a normal mammalian action potential (AP). We investigated this in rabbit ventricular myocytes by examining both the properties of sparks evoked by APs and the activity of LCCs. We measured Ca2+ sparks evoked by repeated APs with pipettes containing 2 mmol/L EGTA and single LCC activity in cell-attached patches depolarized to +50 mV using pipettes containing 110 mmol/L Ba2+.