camera-based-measurements-intracellular-na-murine-atrial

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the ...

University of Maryland School of Medicine

Atrial fibrillation (AF) is characterized by sustained high atrial activation rates and arrhythmogenic cellular Ca2+ signaling instability; however, it is not clear how a high atrial rate and Ca2+ instability may be related. Here, we characterized subcellular Ca2+ signaling after 5 days of high atrial rates in a rabbit model. While some changes were similar to those in persistent AF, we identified a distinct pattern of stabilized subcellular Ca2+ signaling. Ca2+ sparks, arrhythmogenic Ca2+ waves, sarcoplasmic reticulum (SR) Ca2+ leak, and SR Ca2+ content were largely unaltered. Based on computational analysis, these findings were consistent with a higher Ca2+ leak due to PKA-dependent phosphorylation of SR Ca2+ channels (RyR2s), fewer RyR2s, and smaller RyR2 clusters in the SR. We determined that less Ca2+ release per [Ca2+]i transient, increased Ca2+ buffering strength, shortened action potentials, and reduced L-type Ca2+ current contribute to a stunning reduction of intracellular Na+ concentration following rapid atrial pacing. In both patients with AF and in our rabbit model, this silencing led to failed propagation of the [Ca2+]i signal to the myocyte center. We conclude that sustained high atrial rates alone silence Ca2+ signaling and do not produce Ca2+ signaling instability, consistent with an adaptive molecular and cellular response to atrial tachycardia. 

Tachycardia-induced silencing of subcellular Ca2+ signaling in atrial myocytes.

Subcellular calcium signalling silencing is a novel and distinct cellular and molecular adaptive response to rapid cardiac activation. Calcium signalling silencing develops during short-term sustained rapid atrial activation as seen clinically during paroxysmal atrial fibrillation (AF). It is the first 'anti-arrhythmic' adaptive response in the setting of AF and appears to counteract the maladaptive changes that lead to intracellular Ca signalling instability and Ca -based arrhythmogenicity. Calcium signalling silencing results in a failed propagation of the [Ca ] signal to the myocyte centre both in patients with AF and in a rabbit model. This adaptive mechanism leads to a substantial reduction in the expression levels of calcium release channels (ryanodine receptors, RyR2) in the sarcoplasmic reticulum, and the frequency of Ca sparks and arrhythmogenic Ca waves remains low. Less Ca release per [Ca ] transient, increased fast Ca buffering strength, shortened action potentials and reduced L-type Ca current contribute to a substantial reduction of intracellular [Na ]. These features of Ca signalling silencing are distinct and in contrast to the changes attributed to Ca -based arrhythmogenicity. Some features of Ca signalling silencing prevail in human AF suggesting that the Ca signalling 'phenotype' in AF is a sum of Ca stabilizing (Ca signalling silencing) and Ca destabilizing (arrhythmogenic unstable Ca signalling) factors. Calcium signalling silencing is a part of the mechanisms that contribute to the natural progression of AF and may limit the role of Ca -based arrhythmogenicity after the onset of AF.

Calcium signalling silencing in atrial fibrillation.

A gentle optical examination of the mitochondrial permeability transition pore (mPTP) opening events was carried out in isolated quiescent ventricular myocytes by tracking the inner membrane potential (ΔΨ) using TMRM (tetramethylrhodamine methyl ester). Zeiss Airyscan 880 ″super-resolution" or "high-resolution" imaging was done with very low levels of illumination (0.009% laser power). In cellular areas imaged every 9 s (ROI or regions of interest), transient depolarizations of variable amplitudes occurred at increasing rates for the first 30 min. The time to first depolarization events was 8.4 min (±1.1 SEM n = 21 cells). At longer times, essentially permanent and irreversible depolarizations occurred at an increasing fraction of all events. In other cellular areas surrounding the ROI, mitochondria were rarely illuminated (once per 5 min) and virtually no permanent depolarization events occurred for over 1 h of imaging. These findings suggest that photon stress due to the imaging itself plays an important role in the generation of both the transient mPTP opening events as well as the permanent mPTP opening events. Consistent with the evidence that photon "stress" in mitochondria loaded with virtually any photon absorbing substance, generates reactive oxygen species (ROS) [1-5], we show that cyclosporine-A (CsA, 10 μM) and the antioxidant n-acetyl cysteine (NAC, 10 mM), reduced the number of events by 80% and 93% respectively. Furthermore, CsA and NAC treatment led to the virtual disappearance of permanent depolarization events. Nevertheless, all transient depolarization events in any condition (control, CsA and NAC) appeared to repolarize with a similar half-time of 30 ± 6 s (n = 478) at 37 °C. Further experiments showed quantitatively similar results in cerebral vascular smooth muscle cells, using a different confocal system, and different photon absorbing reagent (TMRE; tetramethylrhodamine ethyl ester). In these experiments, using modest power (1% laser power) transient depolarization events were seen in only 8 out of 23 cells while with higher power (8%), all cells showed transient events, which align with the level of photon stress being the driver of the effect. Together, our findings suggest that photon-induced ROS is sufficient to cause depolarization events of individual mitochondria in quiescent cells; without electrical or mechanical activity to stimulates mitochondrial metabolism, and without raising the mitochondrial matrix Ca. In a broad context, these findings neither support nor deny the relevance or occurrence of ΔΨ depolarization events in specific putatively physiologic mitochondrial behaviors such as MitoFlashes [6,7] or MitoWinks [8]. Instead, our findings raise a caution with regards to the physiological and pathophysiological functions attributed to singular ΔΨ depolarization events when those functions are investigated using photon absorbing substances. Nevertheless, using photon stress as a tool ("Optical Stress-Probe"), we can extract information on the activation, reversibility, permanency and kinetics of mitochondrial depolarization. These data may provide new information on mPTP, help identify the mPTP protein complex, and establish the physiological function of the mPTP protein complex and their links to MitoFlashes and MitoWinks.

Dynamics of the mitochondrial permeability transition pore: Transient and permanent opening events.

Ca flux into the mitochondrial matrix through the MCU holocomplex (MCU) has recently been measured quantitatively and with milliseconds resolution for the first time under physiological conditions in both heart and skeletal muscle. Additionally, the dynamic levels of Ca in the mitochondrial matrix ([Ca]) of cardiomyocytes were measured as it was controlled by the balance between influx of Ca into the mitochondrial matrix through MCU and efflux through the mitochondrial Na / Ca exchanger (NCLX). Under these conditions [Ca] was shown to regulate ATP production by the mitochondria at only a few critical sites. Additional functions attributed to [Ca] continue to be reported in the literature. Here we review the new findings attributed to MCU function and provide a framework for understanding and investigating mitochondrial Ca influx features, many of which remain controversial. The properties and functions of the MCU subunits that constitute the holocomplex are challenging to tease apart. Such distinct subunits include EMRE, MCUR1, MICUx (i.e. MICU1, MICU2, MICU3), and the pore-forming subunits (MCU). Currently, the specific set of functions of each subunit remains non-quantitative and controversial. The more contentious issues are discussed in the context of the newly measured native MCU Ca flux from heart and skeletal muscle. These MCU Ca flux measurements have been shown to be a highly-regulated, tissue-specific with femto-Siemens Ca conductances and with distinct extramitochondrial Ca ([Ca]) dependencies. These data from cardiac and skeletal muscle mitochondria have been examined quantitatively for their threshold [Ca] levels and for hypothesized gatekeeping function and are discussed in the context of model cell (e.g. HeLa, MEF, HEK293, COS7 cells) measurements. Our new findings on MCU dependent matrix [Ca] signaling provide a quantitative basis for on-going and new investigations of the roles of MCU in cardiac function ranging from metabolic fuel selection, capillary blood-flow control and the pathological activation of the mitochondrial permeability transition pore (mPTP). Additionally, this review presents the use of advanced new methods that can be readily adapted by any investigator to enable them to carry out quantitative Ca measurements in mitochondria while controlling the inner mitochondrial membrane potential, ΔΨ.

Calcium influx through the mitochondrial calcium uniporter holocomplex, MCU.

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia, affecting more than 33 million people worldwide. Despite important advances in therapy, AF's incidence remains high, and treatment often results in recurrence of the arrhythmia. A better understanding of the cellular and molecular changes that (1) trigger AF and (2) occur after the onset of AF will help to identify novel therapeutic targets. Over the past 20 years, a large body of research has shown that intracellular Ca handling is dramatically altered in AF. While some of these changes are arrhythmogenic, other changes counteract cellular arrhythmogenic mechanisms (Calcium Signaling Silencing). The intracellular Na concentration ([Na]) is a key regulator of intracellular Ca handling in cardiac myocytes. Despite its importance in the regulation of intracellular Ca handling, little is known about [Na], its regulation, and how it might be changed in AF. Previous work suggests that there might be increases in the late component of the atrial Na current (I) in AF, suggesting that [Na] levels might be high in AF. Indeed, a pharmacological blockade of I has been suggested as a treatment for AF. Here, we review calcium signaling silencing and changes in intracellular Na homeostasis during AF. We summarize the proposed arrhythmogenic mechanisms associated with increases in I during AF and discuss the evidence from clinical trials that have tested the pharmacological I blocker ranolazine in the treatment of AF.

Calcium Signaling Silencing in Atrial Fibrillation: Implications for Atrial Sodium Homeostasis.

Mechanistically driven therapies for atrial fibrillation (AF), the most common cardiac arrhythmia, are urgently needed, the development of which requires improved understanding of the cellular signaling pathways that facilitate the structural and electrophysiological remodeling that occurs in the atria. Similar to humans, increased persistent Na+ current leads to the development of an atrial myopathy and spontaneous and long-lasting episodes of AF in mice. How increased persistent Na+ current causes both structural and electrophysiological remodeling in the atria is unknown. We crossbred mice expressing human F1759A-NaV1.5 channels with mice expressing human mitochondrial catalase (mCAT). Increased expression of mCAT attenuated mitochondrial and cellular reactive oxygen species (ROS) and the structural remodeling that was induced by persistent F1759A-Na+ current. Despite the heterogeneously prolonged atrial action potential, which was unaffected by the reduction in ROS, the incidences of spontaneous AF, pacing-induced after-depolarizations, and AF were substantially reduced. Expression of mCAT markedly reduced persistent Na+ current-induced ryanodine receptor oxidation and dysfunction. In summary, increased persistent Na+ current in atrial cardiomyocytes, which is observed in patients with AF, induced atrial enlargement, fibrosis, mitochondrial dysmorphology, early after-depolarizations, and AF, all of which can be attenuated by resolving mitochondrial oxidative stress.

Article	Year
Tachycardia-induced silencing of subcellular Ca2+ signaling in atrial myocytes. The Journal of clinical investigation\| PubMed ID: 25329692	2014
Calcium signalling silencing in atrial fibrillation. The Journal of physiology\| PubMed ID: 28332202	2017
Dynamics of the mitochondrial permeability transition pore: Transient and permanent opening events. Archives of biochemistry and biophysics\| PubMed ID: 30930285	2019
Calcium influx through the mitochondrial calcium uniporter holocomplex, MCU. Journal of molecular and cellular cardiology\| PubMed ID: 33147447	2021
Calcium Signaling Silencing in Atrial Fibrillation: Implications for Atrial Sodium Homeostasis. International journal of molecular sciences\| PubMed ID: 34638854	2021
Attenuating persistent sodium current-induced atrial myopathy and fibrillation by preventing mitochondrial oxidative stress. JCI insight\| PubMed ID: 34710060	2021

Maura Greiser

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