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This article reviews how to analyze data from experiments designed to compare the cellular physiology of two or more groups of animals or people. This is commonly done by measuring data from several cells from each animal and using simple t tests or ANOVA to compare between groups. I use simulations to illustrate that this method can give erroneous positive results by assuming that the cells from each animal are independent of each other. This problem, which may be responsible for much of the lack of reproducibility in the literature, can be easily avoided by using a hierarchical, nested statistics approach., (© 2021 Eisner.)

Calcium ions (Ca 2+ ) play a major role in the cardiac excitation-contraction coupling. Intracellular Ca 2+ concentration increases during systole and falls in diastole thereby determining cardiac contraction and relaxation. Normal cardiac function also requires perfect organization of the ion currents at the cellular level to drive action potentials and to maintain action potential propagation and electrical homogeneity at the tissue level. Any imbalance in Ca 2+ homeostasis of a cardiac myocyte can lead to electrical disturbances. This review aims to discuss cardiac physiology and pathophysiology from the elementary membrane processes that can cause the electrical instability of the ventricular myocytes through intracellular Ca 2+ handling maladies to inherited and acquired arrhythmias. Finally, the paper will discuss the current therapeutic approaches targeting cardiac arrhythmias., (Copyright © 2020 Kistamás, Veress, Horváth, Bányász, Nánási and Eisner.)

Obesity-related hypertension is one of the world's leading causes of death and yet little is understood as to how it develops. As a result, effective targeted therapies are lacking and pharmacological treatment is unfocused. To investigate underlying microvascular mechanisms, we studied small artery dysfunction in a high fat-fed mouse model of obesity. Pressure-induced constriction and responses to endothelial and vascular smooth muscle agonists were studied using myography; the corresponding intracellular Ca 2+ signaling pathways were examined using confocal microscopy. Principally, we observed that the enhanced basal tone of mesenteric resistance arteries was due to failure of intraluminal pressure-induced Ca 2+ spark activation of the large conductance Ca 2+ activated K + potassium channel (BK) within vascular smooth muscle cells. Specifically, the uncoupling site of this mechanotransduction pathway was at the sarcoplasmic reticulum, distal to intraluminal pressure-induced oxidation of Protein Kinase G. In contrast, the vasodilatory function of the endothelium and the underlying endothelial IP-3 and TRPV4 (vanilloid 4 transient receptor potential ion channel) Ca 2+ signaling pathways were not affected by the high-fat diet or the elevated blood pressure. There were no structural alterations of the arterial wall. Our work emphasizes the importance of the intricate cellular pathway by which intraluminal pressure maintains Ca 2+ spark vasoregulation in the origin of obesity-related hypertension and suggests previously unsuspected avenues for pharmacological intervention.

Normal cardiac function requires that intracellular Ca 2+ concentration be reduced to low levels in diastole so that the ventricle can relax and refill with blood. Heart failure is often associated with impaired cardiac relaxation. Little, however, is known about how diastolic intracellular Ca 2+ concentration is regulated. This article first discusses the reasons for this ignorance before reviewing the basic mechanisms that control diastolic intracellular Ca 2+ concentration. It then considers how the control of systolic and diastolic intracellular Ca 2+ concentration is intimately connected. Finally, it discusses the changes that occur in heart failure and how these may result in heart failure with preserved versus reduced ejection fraction.

Changes of intracellular Ca 2+ concentration regulate many aspects of cardiac myocyte function. About 99% of the cytoplasmic calcium in cardiac myocytes is bound to buffers, and their properties will therefore have a major influence on Ca 2+ signaling. This article considers the fundamental properties and identities of the buffers and how to measure them. It reviews the effects of buffering on the systolic Ca 2+ transient and how this may change physiologically, and in heart failure and both atrial and ventricular arrhythmias, as well. It is concluded that the consequences of this strong buffering may be more significant than currently appreciated, and a fuller understanding is needed for proper understanding of cardiac calcium cycling and contractility.

Background Atrial fibrillation ( AF ) is common in the elderly, but rare in the young; however, the changes that occur with age that promote AF are not fully understood. Action potential ( AP ) alternans may be involved in the initiation of AF . Using a translationally relevant model, we investigated whether age-associated atrial vulnerability to AF was associated with susceptibility to AP alternans. Methods and Results AF was induced in conscious young and old sheep using 50 Hz burst pacing. Old sheep were more vulnerable to AF . Monophasic and cellular AP s were recorded from the right atrium in vivo and from myocytes isolated from the left and right atrial appendages. AP alternans occurred at lower stimulation frequencies in old sheep than young in vivo (old, 3.0±0.1 Hz; young, 3.3±0.1 Hz; P<0.05) and in isolated myocytes (old, 1.6±0.1 Hz; young, 2.0±0.1 Hz; P<0.05). Simultaneous recordings of [Ca 2+ ] i and membrane potential in myocytes showed that alternans of AP s and [Ca 2+ ] i often occurred together. However, at low stimulation rates [Ca 2+ ] i alternans could occur without AP alternans, whereas at high stimulation rates AP alternans could still be observed despite disabling Ca 2+ cycling using thapsigargin. Conclusions We have shown, for the first time in a large mammalian model, that aging is associated with increased duration of AF and susceptibility to AP alternans. We suggest that instabilities in Ca 2+ handling initiate alternans at low stimulation rates, but that AP restitution alone can sustain alternans at higher rates.

Atrial fibrillation (AF) is commonly associated with heart failure. A bidirectional relationship exists between the two-AF exacerbates heart failure causing a significant increase in heart failure symptoms, admissions to hospital and cardiovascular death, while pathological remodeling of the atria as a result of heart failure increases the risk of AF. A comprehensive understanding of the pathophysiology of AF is essential if we are to break this vicious circle. In this review, the latest evidence will be presented showing a fundamental role for calcium in both the induction and maintenance of AF. After outlining atrial electrophysiology and calcium handling, the role of calcium-dependent afterdepolarizations and atrial repolarization alternans in triggering AF will be considered. The atrial response to rapid stimulation will be discussed, including the short-term protection from calcium overload in the form of calcium signaling silencing and the eventual progression to diastolic calcium leak causing afterdepolarizations and the development of an electrical substrate that perpetuates AF. The role of calcium in the bidirectional relationship between heart failure and AF will then be covered. The effects of heart failure on atrial calcium handling that promote AF will be reviewed, including effects on both atrial myocytes and the pulmonary veins, before the aspects of AF which exacerbate heart failure are discussed. Finally, the limitations of human and animal studies will be explored allowing contextualization of what are sometimes discordant results.

Cardiac contractility is regulated by changes in intracellular Ca concentration ([Ca 2+ ] i ). Normal function requires that [Ca 2+ ] i be sufficiently high in systole and low in diastole. Much of the Ca needed for contraction comes from the sarcoplasmic reticulum and is released by the process of calcium-induced calcium release. The factors that regulate and fine-tune the initiation and termination of release are reviewed. The precise control of intracellular Ca cycling depends on the relationships between the various channels and pumps that are involved. We consider 2 aspects: (1) structural coupling: the transporters are organized within the dyad, linking the transverse tubule and sarcoplasmic reticulum and ensuring close proximity of Ca entry to sites of release. (2) Functional coupling: where the fluxes across all membranes must be balanced such that, in the steady state, Ca influx equals Ca efflux on every beat. The remainder of the review considers specific aspects of Ca signaling, including the role of Ca buffers, mitochondria, Ca leak, and regulation of diastolic [Ca 2+ ] i ., (© 2017 The Authors.)

The microcirculation is the site of gas and nutrient exchange. Control of central or local signals acting on the myocytes, pericytes and endothelial cells within it, is essential for health. Due to technical problems of accessibility, the mechanisms controlling Ca2+ signalling and contractility of myocytes and pericytes in different sections of microvascular networks in situ have not been investigated. We aimed to investigate Ca2+ signalling and functional responses, in a microcirculatory network in situ. Using live confocal imaging of ureteric microvascular networks, we have studied the architecture, morphology, Ca2+ signalling and contractility of myocytes and pericytes. Ca2+ signals vary between distributing arcade and downstream transverse and precapillary arterioles, are modified by agonists, with sympathetic agonists being ineffective beyond transverse arterioles. In myocytes and pericytes, Ca2+ signals arise from Ca2+ release from the sarcoplasmic reticulum through inositol 1,4,5-trisphosphate-induced Ca2+ release and not via ryanodine receptors or Ca2+ entry into the cell. The responses in pericytes are less oscillatory, slower and longer-lasting than those in myocytes. Myocytes and pericytes are electrically coupled, transmitting Ca2+ signals between arteriolar and venular networks dependent on gap junctions and Ca2+ entry via L-type Ca2+ channels. Endothelial Ca2+ signalling inhibits intracellular Ca2+ oscillations in myocytes and pericytes via L-arginine/nitric oxide pathway and intercellular propagating Ca2+ signals via EDHF. Increases of Ca2+ in pericytes and myocytes constrict all vessels except capillaries. These data reveal the structural and signalling specializations allowing blood flow to be regulated by myocytes and pericytes., (Copyright © 2013 The Authors. Published by Elsevier India Pvt Ltd. All rights reserved.)

Rationale: mutations of the ryanodine receptor (RyR) cause catecholaminergic polymorphic ventricular tachycardia (CPVT). These mutations predispose to the generation of Ca waves and delayed afterdepolarizations during adrenergic stimulation. Ca waves occur when either sarcoplasmic reticulum (SR) Ca content is elevated above a threshold or the threshold is decreased. Which of these occurs in cardiac myocytes expressing CPVT mutations is unknown., Objective: we tested whether the threshold SR Ca content is different between control and CPVT and how it relates to SR Ca content during β-adrenergic stimulation., Methods and Results: ventricular myocytes from the RyR2 R4496C(+/-) mouse model of CPVT and wild-type (WT) controls were voltage-clamped; diastolic SR Ca content was measured and compared with the Ca wave threshold. The results showed the following. (1) In 1 mmol/L [Ca(2+)](o), β-adrenergic stimulation with isoproterenol (1μmol/L) caused Ca waves only in R4496C. (2) SR Ca content and Ca wave threshold in R4496C were lower than those in WT. (3) β-Adrenergic stimulation increased SR Ca content by a similar amount in both R4496C and WT. (4) β-Adrenergic stimulation increased the threshold for Ca waves. (5) During β-adrenergic stimulation in R4496C, but not WT, the increase of SR Ca was sufficient to reach threshold and produce Ca waves., Conclusions: in the R4496C CPVT model, the RyR is leaky, and this lowers both SR Ca content and the threshold for waves. β-Adrenergic stimulation produces Ca waves by increasing SR Ca content and not by lowering threshold.

Elevations in reactive oxygen species are implicated in many disease states and cause systolic and diastolic myocardial dysfunction. To understand the underlying cellular dysfunction, we characterised the effects of H₂O₂ on [Ca(2+)](i) handling and contractility in the rat ventricular myocyte. This was achieved using patch clamping, [Ca(2+)](i) measurement using Fluo-3, video edge detection and confocal microscopy. All experiments were performed at 37°C. 200 μM H₂O₂ resulted in a 44% decrease in the [Ca(2+)](i) transient amplitude, a 30% increase in diastolic [Ca(2+)](i) and an 18% decrease in the rate of systolic Ca(2+) removal. This was associated with a 61% reduction in systolic shortening, a contracture of 3 μm and a 42% increase in relaxation time respectively. The decrease in the [Ca(2+)](i) transient amplitude could be explained by a 27% decrease in SR Ca(2+) content. This, in turn results from a 22% decrease of SERCA activity. The decreased SR Ca(2+) content also provides a mechanism for a reduction in [Ca(2+)](i) spark frequency with no evidence for a Ca(2+) independent modification of ryanodine receptor open probability. We conclude that decreased SERCA activity is the major factor responsible for the changes of the systolic [Ca(2+)](i) transient., (Copyright © 2010 Elsevier Ltd. All rights reserved.)