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Heart Failure with preserved ejection fraction (HFpEF) has a high rate of sudden cardiac death (SCD) and empirical treatment is ineffective. We developed a novel preclinical model of metabolic HFpEF that presents with stress-induced ventricular tachycardia (VT). Mechanistically, we discovered arrhythmogenic changes in intracellular Ca 2+ handling distinct from the changes pathognomonic for heart failure with reduced ejection fraction. We further show that dantrolene, a stabilizer of the ryanodine receptor Ca 2+ channel, attenuates HFpEF-associated arrhythmogenic Ca 2+ handling in vitro and suppresses stress-induced VT in vivo. We propose ryanodine receptor stabilization as a mechanistic approach to mitigation of malignant VT in metabolic HFpEF., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Ca 2+ flux into the mitochondrial matrix through the MCU holocomplex (MCU cx ) 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 2+ in the mitochondrial matrix ([Ca 2+ ] m ) of cardiomyocytes were measured as it was controlled by the balance between influx of Ca 2+ into the mitochondrial matrix through MCU cx and efflux through the mitochondrial Na + / Ca 2+ exchanger (NCLX). Under these conditions [Ca 2+ ] m was shown to regulate ATP production by the mitochondria at only a few critical sites. Additional functions attributed to [Ca 2+ ] m continue to be reported in the literature. Here we review the new findings attributed to MCU cx function and provide a framework for understanding and investigating mitochondrial Ca 2+ influx features, many of which remain controversial. The properties and functions of the MCU cx 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 pore ). 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 cx Ca 2+ flux from heart and skeletal muscle. These MCU cx Ca 2+ flux measurements have been shown to be a highly-regulated, tissue-specific with femto-Siemens Ca 2+ conductances and with distinct extramitochondrial Ca 2+ ([Ca 2+ ] i ) dependencies. These data from cardiac and skeletal muscle mitochondria have been examined quantitatively for their threshold [Ca 2+ ] i 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 cx dependent matrix [Ca 2+ ] m signaling provide a quantitative basis for on-going and new investigations of the roles of MCU cx 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 2+ measurements in mitochondria while controlling the inner mitochondrial membrane potential, ΔΨ m ., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Single-cell metabolic investigations are hampered by the absence of flexible tools to measure local partial pressure of O 2 (pO 2 ) at high spatial-temporal resolution. To this end, we developed an optical sensor capable of measuring local pericellular pO 2 for subcellular resolution measurements with confocal imaging while simultaneously carrying out electrophysiological and/or chemo-mechanical single cell experiments. Here we present the OxySplot optrode, a ratiometric fluorescent O 2 -micro-sensor created by adsorbing O 2 -sensitive and O 2 -insensitive fluorophores onto micro-particles of silica. To protect the OxySplot optrode from the components and reactants of liquid environment without compromising access to O 2 , the micro-particles are coated with an optically clear silicone polymer (PDMS, polydimethylsiloxane). The PDMS coated OxySplot micro-particles are used alone or in a thin (~50 μm) PDMS layer of arbitrary shape referred to as the OxyMat. Additional top coatings on the OxyMat (e.g., fibronectin, laminin, polylysine, special photoactivatable surfaces etc.) facilitate adherence of cells. The OxySplots report the cellular pO 2 and micro-gradients of pO 2 without disrupting the flow of extracellular solutions or interfering with patch-clamp pipettes, mechanical attachments, and micro-superfusion. Since OxySplots and a cell can be imaged and spatially resolved, calibrated changes of pO 2 and intracellular events can be imaged simultaneously. In addition, the response-time (t 0.5  = 0.7 s, 0-160 mmHg) of OxySplots is ~100 times faster than amperometric Clark-type polarization microelectrodes. Two usage example of OxySplots with cardiomyocytes show (1) OxySplots measuring pericellular pO 2 while tetramethylrhodamine methyl-ester (TMRM) was used to measure mitochondrial membrane potential (ΔΨ m ); and (2) OxySplots measuring pO 2 during ischemia and reperfusion while rhod-2 was used to measure cytosolic [Ca 2+ ] i levels simultaneously. The OxySplot/OxyMat optrode system provides an affordable and highly adaptable optical sensor system for monitoring pO 2 with a diverse array of imaging systems, including high-speed, high-resolution confocal microscopes while physiological features are measured simultaneously., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Calcium-induced calcium release is the principal mechanism that triggers the cell-wide [Ca(2+)]i transient that activates muscle contraction during cardiac excitation-contraction coupling (ECC). Here, we characterize this process in mouse cardiac myocytes with a novel mathematical action potential (AP) model that incorporates realistic stochastic gating of voltage-dependent L-type calcium (Ca(2+)) channels (LCCs) and sarcoplasmic reticulum (SR) Ca(2+) release channels (the ryanodine receptors, RyR2s). Depolarization of the sarcolemma during an AP stochastically activates the LCCs elevating subspace [Ca(2+)] within each of the cell's 20,000 independent calcium release units (CRUs) to trigger local RyR2 opening and initiate Ca(2+) sparks, the fundamental unit of triggered Ca(2+) release. Synchronization of Ca(2+) sparks during systole depends on the nearly uniform cellular activation of LCCs and the likelihood of local LCC openings triggering local Ca(2+) sparks (ECC fidelity). The detailed design and true SR Ca(2+) pump/leak balance displayed by our model permits investigation of ECC fidelity and Ca(2+) spark fidelity, the balance between visible (Ca(2+) spark) and invisible (Ca(2+) quark/sub-spark) SR Ca(2+) release events. Excess SR Ca(2+) leak is examined as a disease mechanism in the context of "catecholaminergic polymorphic ventricular tachycardia (CPVT)", a Ca(2+)-dependent arrhythmia. We find that that RyR2s (and therefore Ca(2+) sparks) are relatively insensitive to LCC openings across a wide range of membrane potentials; and that key differences exist between Ca(2+) sparks evoked during quiescence, diastole, and systole. The enhanced RyR2 [Ca(2+)]i sensitivity during CPVT leads to increased Ca(2+) spark fidelity resulting in asynchronous systolic Ca(2+) spark activity. It also produces increased diastolic SR Ca(2+) leak with some prolonged Ca(2+) sparks that at times become "metastable" and fail to efficiently terminate. There is a huge margin of safety for stable Ca(2+) handling within the cell and this novel mechanistic model provides insight into the molecular signaling characteristics that help maintain overall Ca(2+) stability even under the conditions of high SR Ca(2+) leak during CPVT. Finally, this model should provide tools for investigators to examine normal and pathological Ca(2+) signaling characteristics in the heart., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Consumption of adenosine triphosphate (ATP) by the heart can change dramatically as the energetic demands increase from a period of rest to strenuous activity. Mitochondrial ATP production is central to this metabolic response since the heart relies largely on oxidative phosphorylation as its source of intracellular ATP. Significant evidence has been acquired indicating that Ca(2+) plays a critical role in regulating ATP production by the mitochondria. Here the evidence that the Ca(2+) concentration in the mitochondrial matrix ([Ca(2+)]m) plays a pivotal role in regulating ATP production by the mitochondria is critically reviewed and aspects of this process that are under current active investigation are highlighted. Importantly, current quantitative information on the bidirectional Ca(2+) movement across the inner mitochondrial membrane (IMM) is examined in two parts. First, we review how Ca(2+) influx into the mitochondrial matrix depends on the mitochondrial Ca(2+) channel (i.e., the mitochondrial calcium uniporter or MCU). This discussion includes how the MCU open probability (PO) depends on the cytosolic Ca(2+) concentration ([Ca(2+)]i) and on the mitochondrial membrane potential (ΔΨm). Second, we discuss how steady-state [Ca(2+)]m is determined by the dynamic balance between this MCU-based Ca(2+) influx and mitochondrial Na(+)/Ca(2+) exchanger (NCLX) based Ca(2+) efflux. These steady-state [Ca(2+)]m levels are suggested to regulate the metabolic energy supply due to Ca(2+)-dependent regulation of mitochondrial enzymes of the tricarboxylic acid cycle (TCA), the proteins of the electron transport chain (ETC), and the F1F0 ATP synthase itself. We conclude by discussing the roles played by [Ca(2+)]m in influencing mitochondrial responses under pathological conditions. This article is part of a Special Issue entitled "Mitochondria: From BasicMitochondrial Biology to Cardiovascular Disease.", (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The mitochondrial inner membrane has a complex and dynamic structure that plays an important role in the function of this organelle. The internal compartments called cristae are created by processes that are just beginning to be understood. Crista size and morphology influence the internal diffusion of solutes and the surface area of the inner membrane, which is home to critical membrane proteins including ATP synthase and electron transport chain complexes; metabolite and ion transporters including the adenine nucleotide translocase, the calcium uniporter (MCU), and the sodium/calcium exchanger (NCLX); and many more. Here we provide a brief overview of what is known about crista structure and formation, and discuss mitochondrial function in the context of that structure. We also suggest that mathematical modeling of mitochondria that incorporates accurate information about the organelle's internal architecture can lead to a better understanding of its diverse functions. This article is part of a Special Issue entitled 'Calcium Signalling in Heart'., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The free Ca(2+) concentration within the mitochondrial matrix ([Ca(2+)]m) regulates the rate of ATP production and other [Ca(2+)]m sensitive processes. It is set by the balance between total Ca(2+) influx (through the mitochondrial Ca(2+) uniporter (MCU) and any other influx pathways) and the total Ca(2+) efflux (by the mitochondrial Na(+)/Ca(2+) exchanger and any other efflux pathways). Here we review and analyze the experimental evidence reported over the past 40years which suggest that in the heart and many other mammalian tissues a putative Na(+)/Ca(2+) exchanger is the major pathway for Ca(2+) efflux from the mitochondrial matrix. We discuss those reports with respect to a recent discovery that the protein product of the human FLJ22233 gene mediates such Na(+)/Ca(2+) exchange across the mitochondrial inner membrane. Among its many functional similarities to other Na(+)/Ca(2+) exchanger proteins is a unique feature: it efficiently mediates Li(+)/Ca(2+) exchange (as well as Na(+)/Ca(2+) exchange) and was therefore named NCLX. The discovery of NCLX provides both the identity of a novel protein and new molecular means of studying various unresolved quantitative aspects of mitochondrial Ca(2+) movement out of the matrix. Quantitative and qualitative features of NCLX are discussed as is the controversy regarding the stoichiometry of the NCLX Na(+)/Ca(2+) exchange, the electrogenicity of NCLX, the [Na(+)]i dependency of NCLX and the magnitude of NCLX Ca(2+) efflux. Metabolic features attributable to NCLX and the physiological implication of the Ca(2+) efflux rate via NCLX during systole and diastole are also briefly discussed., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

X-ROS signaling is a novel redox signaling pathway that links mechanical stress to changes in [Ca(2+)]i. This pathway is activated rapidly and locally within a muscle cell under physiological conditions, but can also contribute to Ca(2+)-dependent arrhythmia in the heart and to the dystrophic phenotype in the heart and skeletal muscle. Upon physiologic cellular stretch, microtubules serve as mechanotransducers to activate NADPH oxidase 2 in the transverse tubules and sarcolemmal membranes to produce reactive oxygen species (ROS). In the heart, the ROS acts locally to activate ryanodine receptor Ca(2+) release channels in the junctional sarcoplasmic reticulum, increasing the Ca(2+) spark rate and "tuning" excitation-contraction coupling. In the skeletal muscle, where Ca(2+) sparks are not normally observed, the X-ROS signaling process is muted. However in muscular dystrophies, such as Duchenne Muscular Dystrophy and dysferlinopathy, X-ROS signaling operates at a high level and contributes to myopathy. Importantly, Ca(2+) permeable stretch-activated channels are activated by X-ROS and contribute to skeletal muscle pathology. Here we review X-ROS signaling and mechanotransduction in striated muscle, and highlight important questions to drive future work on stretch-dependent signaling. We conclude that X-ROS provides an exciting mechanism for the mechanical control of redox and Ca(2+) signaling, but much work is needed to establish its contribution to physiologic and pathophysiologic processes in diverse cell systems., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Evidence obtained in recent years indicates that, in cardiac myocytes, release of Ca(2+) from the sarcoplasmic reticulum (SR) is regulated by changes in the concentration of Ca(2+) within the SR. In this review, we summarize recent advances in our understanding of this regulatory role, with a particular emphasis on dynamic and local changes in SR [Ca(2+)]. We focus on five important questions that are to some extent unresolved and controversial. These questions concern: (1) the importance of SR [Ca(2+)] depletion in the termination of Ca(2+) release; (2) the quantitative extent of depletion during local release events such as Ca(2+) sparks; (3) the influence of SR [Ca(2+)] refilling on release refractoriness and the propensity for pathological Ca(2+) release; (4) dynamic changes in SR [Ca(2+)] during propagating Ca(2+) waves; and (5) the speed of Ca(2+) diffusion within the SR. With each issue, we discuss data supporting alternative viewpoints, and we identify fundamental questions that are being actively investigated. We conclude with a discussion of experimental and computational advances that will help to resolve controversies. This article is part of a special issue entitled "Local Signaling in Myocytes.", (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Since massive irreversible loss of cardiac myocytes occurs following myocardial injury, injection of human mesenchymal stem cells (hMSCs) has emerged as a promising therapeutic intervention. Despite the growing enthusiasm for this approach, the understanding of how hMSCs evoke cardiac improvement is ever more controversial. The present study critically tests hypothesis that hMSCs provide specific benefit directly to damaged ventricular myocytes. Cultures of neonatal mouse ventricular cardiac myocytes (nMCM) were subjected to two distinct acute stress protocols; incubations with either endotoxin, lipopolysaccharide (LPS) or toxic cytokine, IL-1β. Myocyte injury was assessed in intracellular Ca(2+) signaling assays in fluo-3-loaded nMCMs that were imaged with high temporal resolution by fluorescent microscopy. Following LPS or IL-1β treatment there was profound myocyte injury, manifest by chaotic [Ca(2+)](i) handling, quantified as a 3- to 5-fold increase in spontaneous [Ca(2+)](i) transients. Antibody neutralization experiments reveal such damage is mediated in part by interleukin-18 and not by tumor necrosis factor-α (TNF-α). Importantly, normal [Ca(2+)](i) signaling was preserved when cardiomyocytes were co-cultured with hMSCs. Since normal [Ca(2+)](i) handling was maintained in transwell cultures, where nMCMs and hMSCs were separated by a permeable membrane, a protective paracrine signaling cascade is operable. hMSCs provoke a genetic reprogramming of cardiomyocytes. LPS provokes release of TNFα from nMCMs which is blocked by hMSCs grown in co- or transwell cultures. Consistent with cytokine release, flow cytometry analyses reveal that hMSCs also block the LPS- and IL-1β-dependent activation of cardiac transcription factor, NF-κB. Importantly, hMSC-conditioned medium restores normal Ca(2+) signaling in LPS- and IL-1β-damaged nMCMs. These results reveal new evidence that hMSCs elicit protective and reparative effects on cardiac tissue through molecular reprogramming of the cardiac myocytes themselves. Thus these studies provide novel new insight into the cellular and molecular mechanisms that underlie the therapeutic benefit of hMSCs in the setting of heart failure. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited"., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Cardiac contraction is initiated by the release of Ca(2+) from intracellular stores in response to an action potential, in a process known as "excitation-contraction coupling" (ECC). Here we investigate the maturation of ECC in the rat heart during postnatal development. We provide new information on how proteins of the sarcoplasmic reticulum (SR) and the t-tubules (TTs) assemble to form the structures that support EC coupling during postnatal development. We show that the surface membrane protein, caveolin-3 (Cav3), is a good protein marker for TTs in ventricular myocytes and compared it quantitatively to junctophilin-2 (JP2), a protein found on the SR at sites of SR-TT junctions, or couplons. Although JP2 and Cav3 associate primarily with the SR and TTs, respectively, we found that they occupy the appropriate sites at maturing structures in synchrony, as visualized with high resolution, quantitative 3-dimensional imaging. We also found the surprising result that while both ryanodine receptor type 2, (RyR2) and JP2 proteins are localized to the same membrane and sub-compartments, they assume their positions at very different rates: RyR2 moves to the SR membrane at the Z-disc very early in development while JP2 only appears in the SR membrane as the TTs mature. Our data suggest that, although RyR2 appears to be prepositioned at the sites ultimately occupied by dyad junctions, JP2 arrives at these sites in synchrony with the development of the TTs at the Z-discs. Finally, we report that EC coupling efficiency changes with development, in concert with these structural changes. Thus we provide the first well-integrated information that links the developing organization of proteins underlying EC coupling (RyR2, DHPR, Cav3 and JP2) to the developing efficacy of EC coupling., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Long QT syndrome variant 3 (LQT-3) is a channelopathy in which mutations in SCN5A, the gene coding for the primary heart Na(+) channel alpha subunit, disrupt inactivation to elevate the risk of mutation carriers for arrhythmias that are thought to be calcium (Ca(2+))-dependent. Spontaneous arrhythmogenic diastolic activity has been reported in myocytes isolated from mice harboring the well-characterized Delta KPQ LQT-3 mutation but the link to altered Ca(2+) cycling related to mutant Na(+) channel activity has not previously been demonstrated. Here we have investigated the relationship between elevated sarcoplasmic reticulum (SR) Ca(2+) load and induction of spontaneous diastolic inward current (I(TI)) in myocytes expressing Delta KPQ Na(+) channels, and tested the sensitivity of both to the antianginal compound ranolazine. We combined whole-cell patch clamp measurements, imaging of intracellular Ca(2+), and measurement of SR Ca(2+) content using a caffeine dump methodology. We compared the Ca(2+) content of Delta KPQ(+/-) myocytes displaying I(TI) to those without spontaneous diastolic activity and found that I(TI) induction correlates with higher sarcoplasmic reticulum (SR) Ca(2+). Both spontaneous diastolic I(TI) and underlying Ca(2+) waves are inhibited by ranolazine at concentrations that preferentially target I(NaL) during prolonged depolarization. Furthermore, ranolazine I(TI) inhibition is accompanied by a small but significant decrease in SR Ca(2+) content. Our results provide the first direct evidence that induction of diastolic transient inward current (I(TI)) in Delta KPQ(+/-) myocytes occurs under conditions of elevated SR Ca(2+) load.

The elementary event of Ca(2+) release in heart is the Ca(2+) spark. It occurs at a low rate during diastole, activated only by the low cytosolic [Ca(2+)](i). Synchronized activation of many sparks is due to the high local [Ca(2+)](i) in the region surrounding the sarcoplasmic reticulum (SR) Ca(2+) release channels and is responsible for the systolic [Ca(2+)](i) transient. The biophysical basis of this calcium signaling is discussed. Attention is placed on the local organization of the ryanodine receptors (SR Ca(2+) release channels, RyRs) and the other proteins that underlie and modulate excitation-contraction (EC) coupling. A brief review of specific elements that regulate SR Ca(2+) release (including SR lumenal Ca(2+) and coupled gating of RyRs) is presented. Finally integrative calcium signaling in heart is presented in the context of normal heart function and heart failure.