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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.)

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.

Rationale: In ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI)., Objective: To characterize RyR functional properties in relation to TT proximity, at baseline and after MI., Methods: Myocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca(2+) transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca(2+) release, F>F(50) within 20 ms) or their absence (delayed areas). Spontaneous Ca(2+) release events during diastole, Ca(2+) sparks, reflecting RyR activity and properties, were subsequently assigned to either category., Results: In CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na(+)/Ca(2+) exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca(2+) influx via Ca(2+) channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca(2+) content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca(2+) removal by NCX at the membrane was significantly lower in MI., Conclusion: TT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca(2+) loss and raise SR Ca(2+) content, but may promote Ca(2+) waves.

Rationale: Persistent atrial fibrillation (AF) has been associated with structural and electric remodeling and reduced contractile function., Objective: To unravel mechanisms underlying reduced sarcoplasmic reticulum (SR) Ca(2+) release in persistent AF., Methods: We studied cell shortening, membrane currents, and [Ca(2+)](i) in right atrial myocytes isolated from sheep with persistent AF (duration 129+/-39 days, N=16), compared to matched control animals (N=21). T-tubule density, ryanodine receptor (RyR) distribution, and local [Ca(2+)](i) transients were examined in confocal imaging., Results: Myocyte shortening and underlying [Ca(2+)](i) transients were profoundly reduced in AF (by 54.8% and 62%, P<0.01). This reduced cell shortening could be corrected by increasing [Ca(2+)](i). SR Ca(2+) content was not different. Calculated fractional SR Ca(2+) release was reduced in AF (by 20.6%, P<0.05). Peak Ca(2+) current density was modestly decreased (by 23.9%, P<0.01). T-tubules were present in the control atrial myocytes at low density and strongly reduced in AF (by 45%, P<0.01), whereas the regular distribution of RyR was unchanged. Synchrony of SR Ca(2+) release in AF was significantly reduced with increased areas of delayed Ca(2+) release. Propagation between RyR was unaffected but Ca(2+) release at subsarcolemmal sites was reduced. Rate of Ca(2+) extrusion by Na(+)/Ca(2+) exchanger was increased., Conclusions: In persistent AF, reduced SR Ca(2+) release despite preserved SR Ca(2+) content is a major factor in contractile dysfunction. Fewer Ca(2+) channel-RyR couplings and reduced efficiency of the coupling at subsarcolemmal sites, possibly related to increased Na(+)/Ca(2+) exchanger, underlie the reduction in Ca(2+) release.

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.

In the cardiac dyad, sarcolemmal L-type Ca(2+) channels (LCCs) and sarcoplasmic reticulum (SR) Ca(2+) release channels (RyR) are structurally in close proximity. This organization provides for an efficient functional coupling, tuning SR Ca(2+) release for optimal contraction of the myocyte. Given that LCC are regulated by the prevailing [Ca(2+)], this structural organization is the setting for feedback mechanisms and crosstalk. A defective coupling of Ca(2+) influx via LCC to activation of RyR has been implicated in reduced SR Ca(2+) release in heart failure. Both functional changes in LCC properties and structural re-organization of LCC in T-tubules could be involved. LCC are regulated by cytosolic Ca(2+), and crosstalk with SR Ca(2+) handling occurs on a long-term basis, i.e. during steady-state changes in heart rate, on an intermediate-term basis, i.e. on a beat-to-beat basis during sudden rate changes, and on a very short- or immediate-term basis, i.e. during a single heartbeat. We review the properties and consequences of these different feedback mechanisms and the changes in heart failure and cardiac hypertrophy that have thus far been studied.

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.

Reducing the open probability of the ryanodine receptor (RyR) has been proposed to have beneficial effects in heart failure. We investigated whether conditional FKBP12.6 overexpression at the time of myocardial infarction (MI) could improve cardiac remodelling and cell Ca2+ handling. Wild-type (WT) mice and mice overexpressing FKBP12.6 (Tg) were studied on average 7.5 ± 0.2 weeks after MI and compared with sham-operated mice for in vivo, myocyte function and remodelling. At baseline, unloaded cell shortening in Tg was not different from WT. The [Ca2+]i transient amplitude was similar, but sarcoplasmic reticulum (SR) Ca2+ content was larger in Tg, suggesting reduced fractional release. Spontaneous spark frequency was similar despite the increased SR Ca2+ content, consistent with a reduced RyR channel open probability in Tg. After MI, left ventricular dilatation and myocyte hypertrophy were present in both groups, but more pronounced in Tg. Cell shortening amplitude was unchanged with MI in WT, but increased with MI in Tg. The amplitude of the [Ca2+]i transient was not affected by MI in either genotype, but time to peak was increased; this was most pronounced in Tg. The SR Ca2+ content and Na+- Ca2+ exchanger function were not affected by MI. Spontaneous spark frequency was increased significantly after MI in Tg, and larger than in WT (at 4 Hz, 2.6 ± 0.4 sparks (100 μm)−1 s−1 in Tg MI versus 1.6 ± 0.2 sparks (100 μm)−1 s−1 in WT MI; P < 0.05). We conclude that FKPB12.6 overexpression can effectively reduce RyR open probability with maintained cardiomyocyte contraction. However, this approach appears insufficient to prevent and reduce post-MI remodelling, indicating that additional pathways may need to be targeted. [ABSTRACT FROM AUTHOR]

The article presents a study which discloses the fundamental mechanisms diluted sarcoplasmic reticulum (SR) Ca2+ discharge in persistent atrial fibrillation (AF). The study examines the membrane currents and cell shortening in right atrial myocytes separated from sheep with persistent AF and compared it to matched control animals. Conclusion shows that in persistent AF, diluted SR Ca2+ discharge is a primary factor in contractile dysfunction.

The article discusses a study which examines the effect of the pharmacological inhibition of Na/Ca exchange (NCX) on cellular Ca2+. The study supports the hypothesis that in normal myocytes and physiological conditions, the drug SEA-0400 would not exhibit mode dependence. Even partial inhibition of NCX would lead to a net gain of Ca2+. The study also confirms the potency of SEA-0400 to reduce potentially arrhythmogenic inward NCX currents.

Abstract: Reduced Ca2+ release from the sarcoplasmic reticulum (SR) and a negative force–frequency relation characterize end-stage human heart failure. The MLP–/– mouse with dilated cardiomyopathy is used as a model to explore novel therapeutic interventions but the alterations in Ca2+ handling in MLP–/– remain incompletely understood. We studied [Ca2+]i in left ventricular myocytes from MLP–/– and WT mice (3–4 months old; whole-cell voltage clamp, 30 °C). At 1 Hz stimulation, the amplitude of [Ca2+]i transients was similar. However, in contrast to WT, at higher frequencies the [Ca2+]i transient amplitude declined in MLP–/– and there was no increase in SR Ca2+ content. Unexpectedly, the decline of [Ca2+]i was faster in MLP–/– than in WT (at 1 Hz, τ of 80 ± 9 vs. 174 ± 29 ms, P < 0.001) and the frequency-dependent acceleration of the decline was abolished suggesting an enhanced basal SERCA activity. Indeed, the Ca2+ affinity of SR Ca2+ uptake in homogenates was higher in MLP–/–, with the maximal uptake rate similar to WT. Phosphorylation of phospholamban in MLP–/– was increased (2.3-fold at Ser16 and 2.9-fold at the Thr17 site, P < 0.001) with similar SERCA and total phospholamban protein levels. On increasing stimulation frequency to 4 Hz, WT, but not MLP–/–, myocytes had a net gain of Ca2+, suggesting inadequate Ca2+ sequestration in MLP–/–. In conclusion, increased baseline phosphorylation of phospholamban in MLP–/– leads to a reduced reserve for frequency-dependent increase of Ca2+ release. This represents a novel paradigm for altered Ca2+ handling in heart failure, underscoring the importance of phosphorylation pathways. [Copyright &y& Elsevier]