Calcium signaling underlies much of physiology. Almost all the Ca²⁺ in the cytoplasm is bound to buffers, with typically only ∼1% being freely ionized at resting levels in most cells. Physiological Ca²⁺ buffers include small molecules and proteins, and experimentally Ca²⁺ indicators will also buffer calcium. The chemistry of interactions between Ca²⁺ and buffers determines the extent and speed of Ca²⁺ binding. The physiological effects of Ca²⁺ buffers are determined by the kinetics with which they bind Ca²⁺ and their mobility within the cell. The degree of buffering depends on factors such as the affinity for Ca²⁺, the Ca²⁺ concentration, and whether Ca²⁺ ions bind cooperatively. Buffering affects both the amplitude and time course of cytoplasmic Ca²⁺ signals as well as changes of Ca²⁺ concentration in organelles. It can also facilitate Ca²⁺ diffusion inside the cell. Ca²⁺ buffering affects synaptic transmission, muscle contraction, Ca²⁺ transport across epithelia, and the killing of bacteria. Saturation of buffers leads to synaptic facilitation and tetanic contraction in skeletal muscle and may play a role in inotropy in the heart. This review focuses on the link between buffer chemistry and function and how Ca²⁺ buffering affects normal physiology and the consequences of changes in disease. As well as summarizing what is known, we point out the many areas where further work is required.

Intercellular coupling between ventricular myocytes and myofibroblasts was studied by co‐culturing adult rabbit ventricular myocytes with previously prepared layers of cardiac myofibroblasts. Intercellular coupling was examined by: (i) tracking the movement of the fluorescent dye calcein; (ii) immunostaining for connexin 43 (Cx43); and (iii) measurement of intracellular [Ca²⁺] ([Ca²⁺]_i). The effects of stimulating ventricular myocytes on the underlying myofibroblasts was examined by confocal measurements of [Ca²⁺]_i using fluo‐3. When ventricular myocytes were preloaded with calcein and co‐cultured with myofibroblasts for 24 h, calcein fluorescence was detected in 52 ± 4% (n= 8 co‐cultures) of surrounding myofibroblasts. Treatment with the gap junction uncoupler heptanol significantly reduced the movement of calcein (12 ± 3%, n= 6 co‐cultures). Immunostaining showed expression of Cx43 in co‐cultured myofibroblasts and myocytes. Field stimulation of ventricular myocytes co‐cultured with myofibroblasts increased myofibroblast [Ca²⁺]_i, no response was observed after treatment with heptanol or stimulation of fibroblasts in the absence of ventricular myocytes. Action potential parameters of ventricular myocytes in co‐culture were similar to control values. However, application of the hormone sphingosine‐1‐phosphate (S‐1‐P) to the co‐culture caused a depolarization of ventricular myocytes to approximately −20 mV. Sphingosine‐1‐phosphate had no effect on ventricular myocytes alone. Voltage‐clamp measurements of isolated myofibroblasts indicated that S‐1‐P activated a significant quasi‐linear current with a reversal potential of approximately −40 mV. In conclusion, this study shows that stimulation of the ventricular myocyte influences the intracellular Ca²⁺ of the linked myofibroblast via connexons. These intercellular links also allow the myofibroblasts to influence the electrical activity of the myocyte. This work indicates the nature of the gap junction‐mediated bi‐directional interactions that occur between ventricular myocyte and myofibroblast.

Myocardial cellular electrophysiology and intracellular Ca2+ regulation are altered in heart failure. The extent of these changes may vary within the layers of the ventricular wall. To examine this, cell size, action potential and intracellular Ca2+ transient characteristics (Fura-2) were measured in single cardiac myocytes from sub-epicardial, mid-myocardial, and sub-endocardial regions of the left ventricle of rabbits with heart failure. Myocytes were isolated from animals with heart failure induced by chronic coronary artery ligation and from sham operated controls. Trans-membrane potential was measured using high resistance microelectrodes electrodes (30 M omega; 2 M KC1). Fura-2 was loaded into cells by incubation with the AM form. Subsequent fluorescence measurements were used to measure intracellular Ca2+ concentration at a range of stimulus frequencies. Resting cell length was significantly greater in the heart failure group; approximately 115% of control values in sub-epicardial and mid-myocardial cells, and approximately 108% in sub-endocardial cells. Using criteria described by previous studies on other mammalian hearts, functional M cells were identified by a higher maximum rate of depolarisation and longer action potential duration at 90% repolarisation (APD90) compared to the two other myocyte sub-types. In the heart failure group, APD90 and Ca2+ transient duration (CaD50) were prolonged in sub-epicardial and M cells but shortened in sub-endocardial myocytes. These changes were significant at lower stimulus frequencies, but the relative effect diminished at higher frequencies (3 Hz). Peak systolic [Ca2+] was reduced in sub-epicardial and M cells but increased in sub-endocardial cells in the heart failure group compared to controls. At higher stimulus frequencies, end diastolic Ca2+ levels were lower in sub-epicardial cells but higher in sub-endocardial myocytes of the heart failure group compared with controls. In general, changes were greater in heart failure animals with more severe in vivo ventricular dysfunction (ejection fraction < or = 44%). Heart failure was associated with an increased cell size throughout the left ventricle, but the form of the changes in electrophysiology and Ca2+ transient were dependent on the myocyte sub-type. In particular sub-endocardial cells displayed markedly different changes compared to the other myocyte sub-types.

Human stem cell-derived cardiomyocytes (hSC-CMs) hold great promise as in vitro models to study the electrophysiological effects of novel drug candidates on human ventricular repolarization. Two recent large validation studies have demonstrated the ability of hSC-CMs to detect drug-induced delayed repolarization and "cellrhythmias" (interrupted repolarization or irregular spontaneous beating of myocytes) linked to Torsade-de-Pointes proarrhythmic risk. These (and other) studies have also revealed variability of electrophysiological responses attributable to differences in experimental approaches and experimenter, protocols, technology platforms used, and pharmacologic sensitivity of different human-derived models. Thus, when evaluating drug-induced repolarization effects, there is a need to consider 1) the advantages and disadvantages of different approaches, 2) the need for robust functional characterization of hSC-CM preparations to define "fit for purpose" applications, and 3) adopting standardized best practices to guide future studies with evolving hSC-CM preparations. Examples provided and suggested best practices are instructional in defining consistent, reproducible, and interpretable "fit for purpose" hSC-CM-based applications. Implementation of best practices should enhance the clinical translation of hSC-CM-based cell and tissue preparations in drug safety evaluations and support their growing role in regulatory filings.