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This protocol describes the measurement of Ca(2+) sparks in intact myocytes by using a Ca(2+)-sensitive dye and imaging using laser scanning confocal microscopy. It takes advantage of spontaneous Ca(2+)-release events-sparks-using them as a measure of the activity of ryanodine receptors (RyRs). Two methodologies are described: One requires that cardiomyocytes be stimulated, preferably under voltage clamp by depolarizing pulses, until steady-state is reached, and then stimulation is stopped and Ca(2+) sparks are recorded. The second requires that cells be permeabilized and bathed in a solution to load the cell with Ca(2+) sufficient to elicit Ca(2+) sparks, but not Ca(2+) waves. These are then analyzed offline to quantify spark frequency and morphology. The advantages and disadvantages of each approach are discussed., (© 2015 Cold Spring Harbor Laboratory Press.)

The decline of an intracellular calcium ([Ca(2+)]i) transient during a single excitation-contraction coupling (ECC) cycle reflects the combined activity of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) pump and the sarcolemmal Na(+)-Ca(2+) exchanger (NCX), along with minor contributions of the plasma membrane Ca(2+)-ATPase and mitochondrial Ca(2+) uniporter, in removing Ca(2+) from the cytosol. A traditional approach for assessing the individual components is to fit the decline of the [Ca(2+)]i transient evoked during electrical stimulation with an exponential. This reflects mostly the SERCA-dependent rate of uptake, which can be properly deduced after correcting for a component of NCX removal. As NCX function is an important determinant of the membrane potential as well as the Ca(2+) balance, we present here several detailed protocols for assessing NCX function. As the reversal potential and the amplitudes of the current are highly dependent on the prevailing concentrations of Na(+) and Ca(2+), we show how NCX function can be assessed under highly controlled conditions, with Ca(2+) and Na(+) clamped, as well as under more physiological conditions, with freely changing Ca(2+) and Na(+)., (© 2015 Cold Spring Harbor Laboratory Press.)

In cardiac myocytes, Ca(2+) release from the sarcoplasmic reticulum (SR) Ca(2+) store through the opening of ryanodine receptors (RyRs) is the major source of Ca(2+) for activation of myofilaments and contraction. Over the past 20 years, tools have become available to study this release process in detail, allowing new insights into the regulation of SR Ca(2+) release and RyR function. To assess these processes, we recommend and here review a systematic approach that evaluates the essential transport mechanisms and Ca(2+) fluxes in isolated single cardiac myocytes by using fluorescent Ca(2+) indicators and whole-cell recording of membrane voltage and ionic currents under voltage clamp. The approach includes an assessment of the L-type Ca(2+) current as a trigger for opening of RyRs and release of SR Ca(2+), of the SR Ca(2+) content, of intrinsic properties of RyRs, and of Ca(2+)-removal systems., (© 2015 Cold Spring Harbor Laboratory Press.)

Here, we describe a method for characterizing the L-type Ca(2+) current, ICaL, which is a major trigger for Ca(2+) release from the sarcoplasmic reticulum (SR). The protocol includes measuring ICaL amplitude and voltage dependence and the elicited SR Ca(2+) release. The procedure for measuring ICaL activity is performed using solutions (internal and external) and voltage control such that other ionic currents are eliminated. The resultant relationship between the Ca(2+) current and the associated internal [Ca(2+)]i transient is a first approach for evaluating coupling gain. We discuss which parameters are most appropriate for this analysis and how an evaluation of gain needs to be further explored by measuring the SR Ca(2+) content., (© 2015 Cold Spring Harbor Laboratory Press.)

Here, we describe a protocol for the reliable measurement of the amount of Ca(2+) in the sarcoplasmic reticulum (SR) Ca(2+) store of cardiac myocytes. The whole-cell patch-clamp method is used to provide controlled loading of the SR during conditioning depolarizing pulses, followed by rapid application of a high dose of caffeine to release all SR Ca(2+) and to prevent Ca(2+) reuptake by the SR. Simultaneous measurement of membrane currents records Ca(2+) extruded through the Na(+)-Ca(2+) exchanger. The integral of the caffeine-induced Na(+)-Ca(2+) exchange current is then used as a measure of the SR Ca(2+). Derived measurements include the Ca(2+) buffering capacity and measurement of fractional release as an indicator of coupling gain. Caveats, advantages, and disadvantages of this method and alternative methods are discussed., (© 2015 Cold Spring Harbor Laboratory Press.)

In cardiac myocytes, the physiological increase of intracellular calcium, the [Ca(2+)]i transient, elicited during excitation-contraction coupling typically reaches a peak amplitude of up to 1 µm, from a resting value of ∼100 nm, within 50-100 msec, depending on the species. Various conditions will affect the amplitude and rise time of the [Ca(2+)]i transient and, depending on the nature of the Ca(2+) signals under study, a variety of different probes are available for monitoring changes in intracellular Ca(2+). In this protocol, we focus on Fluo-3, which exists in the cytosol in its salt form K5Fluo-3. This form is practically nonfluorescent in the absence of Ca(2+), but the fluorescence increases dramatically on Ca(2+) binding. Although Fluo-3 is a single excitation-emission dye, it has a number of advantages for investigators, including an ideal dissociation constant (Kd) value and high quantum yield, meaning that it can be used at low concentrations that introduce minimal buffering. Here, we describe the basic setup and methodology for recording the global cytosolic [Ca(2+)]i transient with this probe during simultaneous patch-clamp and whole-cell recording of membrane voltage or of ionic currents under voltage clamp., (© 2015 Cold Spring Harbor Laboratory Press.)

The Na(+)/Ca(2+) exchanger (NCX) is a key regulator of intracellular Ca(2+) in cardiac myocytes, predominantly contributing to Ca(2+) removal during the diastolic relaxation process but also modulating excitation-contraction coupling. NCX is preferentially located in the T-tubules and can be close to or within the dyad, where L-type Ca(2+) channels face ryanodine receptors (RyRs), the Ca(2+) release channels of the sarcoplasmic reticulum. However, especially in larger animals, not all RyRs are in dyads or adjacent to T-tubules, and a substantial fraction of Ca(2+) release from the sarcoplasmic reticulum thus occurs at distance from NCX. This chapter deals with the functional consequences of NCX location and how NCX can modulate diastolic and systolic Ca(2+) events. The loss of T-tubules and the effects on RyR function and NCX modulation are explored, as well as quantitative measurement of local Ca(2+) gradients at the level of the dyadic space.

Cardiomyocytes from failing hearts exhibit reduced levels of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) and/or increased activity of the endogenous SERCA inhibitor phospholamban. The resulting reduction in the Ca(2+) affinity of SERCA impairs SR Ca(2+) cycling in this condition. We have previously investigated the physiological impact of increasing the Ca(2+) affinity of SERCA by substituting SERCA2a with the higher affinity SERCA2b pump. When phospholamban was also ablated, these double knockouts (DKO) exhibited a dramatic reduction in total SERCA levels, severe hypertrophy, and diastolic dysfunction. We presently examined the role of cardiomyocyte Ca(2+) homeostasis in both functional and structural remodeling in these hearts. Despite the low SERCA levels in DKO, we observed near-normal Ca(2+) homeostasis with rapid Ca(2+) reuptake even at high Ca(2+) loads and stimulation frequencies. Well-preserved global Ca(2+) homeostasis in DKO was paradoxically associated with marked activation of the Ca(2+)-dependent nuclear factor of activated T-cell-calcineurin pathway known to trigger hypertrophy. No activation of the MAP kinase signaling pathway was detected. These findings suggest that local changes in Ca(2+) homeostasis may play an important signaling role in DKO, perhaps due to reduced microdomain Ca(2+) buffering by SERCA2b. Furthermore, alterations in global Ca(2+) homeostasis can also not explain impaired in vivo diastolic function in DKO. Taken together, our results suggest that normalizing global cardiomyocyte Ca(2+) homeostasis does not necessarily protect against hypertrophy and heart failure development and that excessively increasing SERCA Ca(2+) affinity may be detrimental.

Block of Na/Ca exchange (NCX) has potential therapeutic applications, in particular, if a mode-selective block could be achieved, but also carries serious risks for disturbing the normal Ca2+ balance maintained by NCX. We have examined the effects of partial inhibition of NCX by SEA-0400 (1 or 0.3 micromol/L) in left ventricular myocytes from healthy pigs or mice and from mice with heart failure (MLP-/-). During voltage clamp ramps with [Ca2+](i) buffering, block of reverse mode block was slightly larger than of forward mode (by 25+/-5%, P<0.05). In the absence of [Ca2+](i) buffering and with sarcoplasmic reticulum (SR) fluxes blocked, rate constants for Ca2+ influx and Ca2+ efflux were reduced to the same extent (to 36+/-6% and 32+/-4%, respectively). With normal SR function the reduction of inward NCX current (I(NCX)) was 57+/-10% (n=10); during large caffeine-induced Ca2+ transients, it was larger (82+/-3%). [Ca2+](i) transients evoked during depolarizing steps increased (from 424+/-27 to 994+/-127 nmol/L at +10 mV, P<0.05), despite a reduction of I(CaL) by 27%. Resting [Ca2+](i) increased; there was a small decrease in the rate of decline of [Ca2+](i). SR Ca2+) content increased more than 2-fold. Contraction amplitude of field-stimulated myocytes increased in healthy myocytes but not in myocytes from MLP-/- mice, in which SR Ca2+ content remained unchanged. These data provide proof-of-principle that even partial inhibition of NCX results in a net gain of Ca2+. Further development of NCX blockers, in particular, for heart failure, must balance potential benefits of I(NCX) reduction against effects on Ca2+ handling by refining mode dependence and/or including additional targets.

In ventricular cardiac myocytes, T-tubule density is an important determinant of the synchrony of sarcoplasmic reticulum (SR) Ca2+ release and could be involved in the reduced SR Ca2+ release in ischemic cardiomyopathy. We therefore investigated T-tubule density and properties of SR Ca2+ release in pigs, 6 weeks after inducing severe stenosis of the circumflex coronary artery (91+/-3%, N=13) with myocardial infarction (8.8+/-2.0% of total left ventricular mass). Severe dysfunction in the infarct and adjacent myocardium was documented by magnetic resonance and Doppler myocardial velocity imaging. Myocytes isolated from the adjacent myocardium were compared with myocytes from the same region in weight-matched control pigs. T-tubule density quantified from the di-8-ANEPPS (di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate) sarcolemmal staining was decreased by 27+/-7% (P<0.05). Synchrony of SR Ca2+ release (confocal line scan images during whole-cell voltage clamp) was reduced in myocardium myocytes. Delayed release (ie, half-maximal [Ca2+]i occurring later than 20 ms) occurred at 35.5+/-6.4% of the scan line in myocardial infarction versus 22.7+/-2.5% in control pigs (P<0.05), prolonging the time to peak of the line-averaged [Ca2+]i transient (121+/-9 versus 102+/-5 ms in control pigs, P<0.05). Delayed release colocalized with regions of T-tubule rarefaction and could not be suppressed by activation of protein kinase A. The whole-cell averaged [Ca2+]i transient amplitude was reduced, whereas L-type Ca2+ current density was unchanged and SR content was increased, indicating a reduction in the gain of Ca2+-induced Ca2+ release. In conclusion, reduced T-tubule density during ischemic remodeling is associated with reduced synchrony of Ca2+ release and reduced efficiency of coupling Ca2+ influx to Ca2+ release.

Torsades de pointes (TdP) ventricular tachycardia typically occurs in the setting of early afterdepolarizations; it contributes to arrhythmias and sudden death in congenital and acquired heart disease. Window L-type Ca2+ current (ICaL) has a central role in the arrhythmogenesis and may be particularly important under beta-adrenergic stimulation. We studied the properties of ICaL in myocytes from the dog with chronic atrioventricular block (cAVB) that has cardiac hypertrophy and an increased susceptibility to TdP. Peak ICaL densities at baseline (K+ - and Na+ -free solutions, 10 mmol l(-1) [EGTA]pip) in cAVB were comparable to control, but inactivation was shifted to the right, resulting in a larger window current area in cAVB. Under beta-adrenergic stimulation, the window current area was increased and shifted to the left, but less so in cAVB (maximum at -27 mV, versus -32 mV in control). ICaL during a step to -35 mV showed a transient reduction immediately after the peak. Test steps to 0 mV, simultaneous recording of [Ca2+]i and manipulation of sarcoplasmic reticulum (SR) Ca2+ release showed that this resulted from inhibition and fast recovery of ICaL with SR Ca2+ release. The extent of this dynamic modulation was larger in cAVB than in control (23 +/- 2% of the initially available current, versus 13 +/- 3%; P<0.05). Early afterdepolarizations (EADs) in cAVB myocytes under beta-adrenergic stimulation typically occurred in the window current voltage range and after decline of [Ca2+]i. In conclusion, in cAVB, the larger window current, its rightward shift and enhanced dynamic modulation by SR Ca2+ release may contribute to an increased incidence of EADs in cAVB under beta-adrenergic stimulation.

KB130015 [KB; 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran] is a novel amiodarone derivative designed to retain the antiarrhythmic effects without the side effects. Unlike amiodarone, KB slows Na(+) current inactivation and could, via an increase in [Na(+)](i), potentially lead to Ca(2+) overload. Therefore, we studied the effects of KB on Na(+) and Ca(2+) handling in single pig ventricular myocytes using the whole-cell ruptured patch-clamp technique and K(5)fluo-3 as [Ca(2+)](i) indicator. KB at 10 microM did not prolong action potential duration but slightly increased the early plateau; spontaneous afterdepolarizations were not observed. The amplitude of the [Ca(2+)](i) transient was larger (434.9 +/- 37.2 versus 326.8 +/- 39.8 nM at baseline, n = 13, P < 0.05), and the time to peak [Ca(2+)](i) was prolonged. During voltage-clamp pulses, [Ca(2+)](i) transient peak was also larger (578.1 +/- 98.9 versus 346.4 +/- 52.6 nM at baseline, P < 0.05). Although L-type Ca(2+) current was reduced (by 21.9% at +10 mV, n = 9, P < 0.05), sarcoplasmic reticulum Ca(2+) content was significantly enhanced with KB. Forward Na(+)/Ca(2+) exchange was significantly decreased after KB application, but reverse mode of the Na(+)/Ca(2+) exchanger was significantly larger, suggesting an increase in [Na(+)](i) with KB. This was confirmed by a 2-fold increase of the [Na(+)]-dependent current generated by the Na/K-ATPase (from 0.17 +/- 0.02 to 0.38 +/- 0.06 pA/pF, P < 0.05). In conclusion, as predicted from the slowing of I(Na) inactivation, KB130015 leads to an increase in [Na(+)](i) and consequently in cellular Ca(2+) load. This effect is partially offset by a decrease in I(CaL) resulting in a mild inotropic effect without the signs of Ca(2+) overload and related arrhythmias usually associated with Na(+) channel openers.

Objectives: During cardiac excitation-contraction coupling, Ca2+ release from the sarcoplasmic reticulum (SR) occurs at the junctional complex with the T-tubules, containing the L-type Ca2+ channels. A partial loss of T-tubules has been described in myocytes from failing canine and human hearts. We examined how graded reduction of T-tubule density would affect the synchrony of Ca2+ release., Methods: Adult pig ventricular myocytes were isolated and cultured for 24 and 72 h. T-tubules, visualized with di-8-ANEPPS, and [Ca2+]i transients (Fluo-3) were recorded during confocal line scan imaging., Results: Cultured cardiomyocytes exhibited a progressive reduction in T-tubule density. [Ca2+]i transients showed small areas of delayed Ca2+ release which gradually increased in number and size with loss of T-tubules. Local [Ca2+]i transients in the delayed regions were reduced. Due to these changes, loss of T-tubules resulted in an overall slowing of the rise of [Ca2+] along the entire line scan and transient magnitude tended to be reduced, but there was no change in SR Ca2+ content. Human myocytes isolated from failing hearts had a T-tubule density comparable to that of freshly isolated pig myocytes. The size, but not the number, of delayed release areas tended to be larger. The overall rate of rise of [Ca2+]i was significantly faster than in pig myocytes with low T-tubule density., Conclusions: Loss of T-tubules reduces the synchrony of SR Ca2+ release. This could contribute to reduced efficiency of excitation-contraction coupling in heart failure, though dyssynchrony in human failing cells appears to be modest.

The [Ca2+]i transient of ventricular myocytes during normal excitation-contraction coupling is the summation of primary Ca2+ release events, which originate at the junction of the sarcoplasmic reticulum (SR) and the T-tubular system. Studies in small mammals have shown a high density of release sites, but little is known of larger mammals. We have studied the spatial distribution of SR Ca2+ release in pig ventricular myocytes using a confocal microscopy. In 69 of 107 cells, large inhomogeneities of Ca2+ release were observed along the longitudinal scan line. Areas where the increase of [Ca2+]i was delayed (time to 50% of peak F/F0 [where F indicates fluorescence intensity, and F0 indicates F at rest] was 26+/-1 ms in delayed areas versus 11+/-2 ms in early areas) and smaller (peak F/F0 was 2.27+/-0.10 for delayed areas versus 2.69+/-0.13 for early areas; n=13 cells, P<0.05) could be up to 26 microm wide. The sum of all delayed areas could make up to 55% of the line scan. The spatial pattern was constant during steady-state stimulation and was not altered by enhancing Ca2+ channel opening or SR Ca2+ content (Bay K8644, isoproterenol). Imaging of sarcolemmal membranes revealed several areas devoid of T tubules, but SR Ca2+ release channels were homogeneously distributed. In contrast, compared with pig myocytes, mouse myocytes had a very dense T-tubular network, no large inhomogeneities of release, and a faster rate of rise of [Ca2+]i. In conclusion, in pig ventricular myocytes, areas of delayed release are related to regional absence of T tubules but not ryanodine receptors. This lower number of functional couplons contributes to a slower overall rate of rise of [Ca2+]i.

Aims: Exercise started early after myocardial infarction (MI) improves in vivo cardiac function and myofilament responsiveness to Ca2+. We investigated whether this represents partial or complete reversal of cellular remodelling. Methods and results: Mice with MI following left coronary ligation were given free access to a running wheel (MIEXE, N = 22) or housed sedentary (MISED, N = 18) for 8 weeks and compared with sedentary sham-operated animals (SHAM, N = 11). Myocytes were enzymatically isolated from the non-infarcted left ventricle. Myocytes in MI were significantly longer and even more so with exercise (165 ± 3 μm in MIEXE vs. 148 ± 3 μm in MISED and 136 ± 2 μm in SHAM; P < 0.05, mean ± SEM); cell width was not different. Contraction was measured during electrical field stimulation at 1, 2, and 4 Hz. Unloaded cell shortening was significantly reduced in MISED (at 1 Hz, L/L0=4.4 ± 0.3% vs. 6.7 ± 0.4% in SHAM; P < 0.05, also at 2 and 4 Hz). Exercise restored cell shortening to SHAM values (MIEXE, L/L0=6.4 ± 0.5%). Membrane currents and [Ca2+]i were measured via whole-cell patch clamping, with Fluo-3 as Ca2+ indicator, all at 30°C. Ca2+ transient amplitude, ICaL and sarcoplasmic reticulum Ca2+ content were not different between the three groups. Diastolic Ca2+ levels at 4 Hz were significantly elevated in MISED only, with a trend to increased spontaneous Ca2+ release events (sparks). Action potential duration was increased and transient outward K+ currents significantly reduced after MI; this was unaffected by exercise. Conclusions: Early voluntary exercise training after MI restores cell contraction to normal values predominantly because of changes in the myofilament Ca2+ response and has a beneficial effect on diastolic Ca2+ handling. However, the beneficial effect is not a complete reversal of remodelling as hypertrophy and loss of repolarizing K+ currents are not affected. [ABSTRACT FROM PUBLISHER]

Objectives: During cardiac excitation–contraction coupling, Ca2+ release from the sarcoplasmic reticulum (SR) occurs at the junctional complex with the T-tubules, containing the L-type Ca2+ channels. A partial loss of T-tubules has been described in myocytes from failing canine and human hearts. We examined how graded reduction of T-tubule density would affect the synchrony of Ca2+ release. Methods: Adult pig ventricular myocytes were isolated and cultured for 24 and 72 h. T-tubules, visualized with di-8-ANEPPS, and [Ca2+]i transients (Fluo-3) were recorded during confocal line scan imaging. Results: Cultured cardiomyocytes exhibited a progressive reduction in T-tubule density. [Ca2+]i transients showed small areas of delayed Ca2+ release which gradually increased in number and size with loss of T-tubules. Local [Ca2+]i transients in the delayed regions were reduced. Due to these changes, loss of T-tubules resulted in an overall slowing of the rise of [Ca2+] along the entire line scan and transient magnitude tended to be reduced, but there was no change in SR Ca2+ content. Human myocytes isolated from failing hearts had a T-tubule density comparable to that of freshly isolated pig myocytes. The size, but not the number, of delayed release areas tended to be larger. The overall rate of rise of [Ca2+]i was significantly faster than in pig myocytes with low T-tubule density. Conclusions: Loss of T-tubules reduces the synchrony of SR Ca2+ release. This could contribute to reduced efficiency of excitation–contraction coupling in heart failure, though dyssynchrony in human failing cells appears to be modest. [Copyright &y& Elsevier]