John H.B. Bridge

Intracellular calcium homeostasis in cardiac myocytes.

Calcium homeostasis in cardiac myocytes results from the integrated function of transsarcolemmal Ca2+ influx and efflux pathways modulated by membrane potential and from intracellular Ca2+ uptake and release caused predominantly by SR function. These processes can be importantly altered in different disease states as well as by pharmacological agents, and the resulting changes in systolic and diastolic [Ca2+]i can cause clinically significant alterations in contraction and relaxation of the heart. It may be anticipated that a rapid increase in our understanding of the pathophysiology of Ca2+ homeostasis in cardiac myocytes will be forthcoming as the powerful new tools of molecular and structural biology are used to investigate the regulation of Ca2+ transport systems.

Enhanced Na+-Ca2+ Exchange in the Infarcted Heart : Implications for Excitation-Contraction Coupling

Cellular Ca2+ regulation is abnormal in diseased hearts. We designed this study to assess the role of the Na(+)-Ca2+ exchanger in excitation-contraction coupling in surviving myocardium of the infarcted heart. We measured cellular contractions and whole-cell currents in single left ventricular myocytes isolated from the hearts of rabbits with healed myocardial infarction (MI). Eight weeks after MI, rabbits had left ventricular dysfunction without overt heart failure. Myocytes isolated from regions adjacent to the infarcted zone were significantly longer than cells from control hearts. At low stimulation rates (0.5 Hz), the amplitude of field-stimulated contractions was increased (11.6 +/- 0.5% versus 10.2 +/- 0.6% resting cell length), whereas the time to peak shortening and action potential duration were prolonged in the MI cells. When stimulation frequency was increased to 2.0 Hz, cellular shortening did not change or decreased in myocytes from infarcted hearts, whereas control cells had a positive shortening-interval relationship. Cells from infarcted hearts had a significantly decreased (31%) L-type Ca2+ current (ICa) density but no change in the current-voltage relationship or the kinetics of ICa inactivation. Maximal Na(+)-Ca2+ exchange current density was significantly increased (32%) in the cells from infarcted hearts. Sarcoplasmic reticulum (SR) Ca2+ content during a stable train of contractions, as estimated from caffeine-induced inward currents, was slightly increased (P = NS) in the MI myocytes. To determine whether Na(+)-Ca2+ exchange influenced SR Ca2+ content, cells were clamped at potentials between -70 and +90 mV for 400 ms. The amplitude of the contraction during a subsequent clamp step to +10 mV was then measured as an index of SR loading that occurred during the preceding clamp step. Steps to positive potentials produced greater augmentation of the subsequent contraction in MI than in control myocytes. In myocytes from the infarcted heart, increased activity of the Na(+)-Ca2+ exchanger may promote Ca2+ entry or decrease Ca2+ extrusion. This relative augmentation of inward Ca2+ flux by the exchanger may enhance SR Ca2+ loading and thus support contractility that would otherwise be impaired as a result of decreased Ca2+ current. However, Ca2+ influx by the exchanger may contribute to the prolongation of contractions in myocytes from infarcted hearts.

Dyssynchronous Ca2+ Sparks in Myocytes From Infarcted Hearts

The kinetics of contractions and Ca2+ transients are slowed in myocytes from failing hearts. The mechanisms accounting for these abnormalities remain unclear. Myocardial infarction (MI) was produced by ligation of the circumflex artery in rabbits. We used confocal microscopy to record spatially resolved Ca2+ transients during field stimulation in left ventricular (LV) myocytes from control and infarcted hearts (3 weeks). Compared with controls, Ca2+ transients in myocytes adjacent to the infarct had lower peak amplitudes and prolonged time courses. Control myocytes showed relatively uniform changes in [Ca2+] throughout the cell after electrical stimulation. In contrast, in MI myocytes [Ca2+] increased inhomogeneously and localized increases in [Ca2+] occurred throughout the rising and falling phases of the Ca2+ transient. Ca2+ content of the sarcoplasmic reticulum did not differ between MI and control myocytes. Peak L-type Ca2+ current density was reduced in MI myocytes. The macroscopic gain function was not different in control and MI myocytes when calculated as the amplitude of the Ca2+ transient/peak ICa. However, when calculated as the peak rate of rise of the Ca2+ transient/peak ICa, the gain function was modestly decreased in the MI myocytes. Application of isoproterenol (100 nmol/L) improved the synchronization of Ca2+ release in MI myocytes at both 0.5 and 1 Hz. The poorly coordinated production of Ca2+ sparks in myocytes from infarcted rabbit hearts likely contributes to the diminished and slowed macroscopic Ca2+ transient. These abnormalities can be largely overcome when phosphorylation of Ca2+ cycling proteins is enhanced by &bgr;-adrenergic stimulation.

Properties of Ca2+ sparks evoked by action potentials in mouse ventricular myocytes

1 Calcium sparks were examined in enzymatically dissociated mouse cardiac ventricular cells using the calcium indicator fluo‐3 and confocal microscopy. The properties of the mouse cardiac calcium spark are generally similar to those reported for other species. 2 Examination of the temporal relationship between the action potential and the time course of calcium spark production showed that calcium sparks are more likely to occur during the initial repolarization phase of the action potential. The latency of their occurrence varied by less than 1·4 ms (s.d.) and this low variability may be explained by the interaction of the gating of L‐type calcium channels with the changes in driving force for calcium entry during the action potential. 3 When fixed sites within the cell are examined, calcium sparks have relatively constant amplitude but the amplitude of the sparks was variable among sites. The low variability of the amplitude of the calcium sparks suggests that more than one sarcoplasmic reticulum (SR) release channel must be involved in their genesis. Noise analysis (with the assumption of independent gating) suggests that > 18 SR calcium release channels may be involved in the generation of the calcium spark. At a fixed site, the response is close to ‘all‐or‐none’ behaviour which suggests that calcium sparks are indeed elementary events underlying cardiac excitation‐contraction coupling. 4 A method for selecting spark sites for signal averaging is presented which allows the time course of the spark to be examined with high temporal and spatial resolution. Using this method we show the development of the calcium spark at high signal‐to‐noise levels.

RELATION BETWEEN REVERSE SODIUM-CALCIUM EXCHANGE AND SARCOPLASMIC RETICULUM CALCIUM RELEASE IN GUINEA PIG VENTRICULAR CELLS

Exchange-inhibitory peptide (XIP) can inhibit sodium-calcium exchange without inhibiting L-type calcium current (ICa). We therefore used this compound to test the hypothesis that reverse sodium-calcium exchange can trigger contraction in guinea pig ventricular myocytes. When cells were dialyzed with 20 mmol/L sodium, rapid blockade of ICa with nifedipine had little effect on cell shortening. However, if reverse exchange was inhibited by first dialyzing the cells with XIP, blockade of ICa largely inhibited cell shortening. In cells dialyzed with 10 mmol/L sodium, about 51% of the maximum cell shortening remained after ICa was blocked. When both ICa and reverse exchange were significantly inhibited with nifedipine and XIP, only 24% of the cell shortening remained; ie, 27% was XIP inhibitable. Cells dialyzed with solutions deficient in sodium exhibited contractions that were largely dependent on ICa (ie, not XIP inhibitable). If the sarcoplasmic reticulum (SR) was disabled with ryanodine and thapsigargin, reverse exchange could not cause contraction. We therefore conclude that with intact SR, reverse sodium-calcium exchange activates contraction by triggering calcium release from the SR in cells dialyzed with either 10 or 20 mmol/L sodium. A scrambled sequence of XIP, sXIP, caused no measurable effect on contraction.

Subcellular Structures and Function of Myocytes Impaired During Heart Failure Are Restored by Cardiac Resynchronization Therapy

Rationale: Cardiac resynchronization therapy (CRT) is an established treatment for patients with chronic heart failure. However, CRT-associated structural and functional remodeling at cellular and subcellular levels is only partly understood. Objective: To investigate the effects of CRT on subcellular structures and protein distributions associated with excitation-contraction coupling of ventricular cardiomyocytes. Methods and Results: Our studies revealed remodeling of the transverse tubular system (t-system) and the spatial association of ryanodine receptor (RyR) clusters in a canine model of dyssynchronous heart failure (DHF). We did not find this remodeling in a synchronous heart failure model based on atrial tachypacing. Remodeling in DHF ranged from minor alterations in anterior left ventricular myocytes to nearly complete loss of the t-system and dissociation of RyRs from sarcolemmal structures in lateral cells. After CRT, we found a remarkable and almost complete reverse remodeling of these structures despite persistent left ventricular dysfunction. Studies of whole-cell Ca2+ transients showed that the structural remodeling and restoration were accompanied with remodeling and restoration of Ca2+ signaling. Conclusions: DHF is associated with regional remodeling of the t-system. Myocytes undergo substantial structural and functional restoration after only 3 weeks of CRT. The finding suggests that t-system status can provide an early marker of the success of this therapy. The results could also guide us to an understanding of the loss and remodeling of proteins associated with the t-system. The steep relationship between free Ca2+ and contraction suggests that some restoration of Ca2+ release units will have a disproportionately large effect on contractility.

Ca2+ Sparks in Rabbit Ventricular Myocytes Evoked by Action Potentials Involvement of Clusters of L-Type Ca2+ Channels

Abstract— It is not clear how many L-type Ca2+ channels (LCCs) are required to ensure that a Ca2+ spark is triggered during a normal mammalian action potential (AP). We investigated this in rabbit ventricular myocytes by examining both the properties of sparks evoked by APs and the activity of LCCs. We measured Ca2+ sparks evoked by repeated APs with pipettes containing 2 mmol/L EGTA and single LCC activity in cell-attached patches depolarized to +50 mV using pipettes containing 110 mmol/L Ba2+. With 2 mmol/L Ca2+ in the external solution, we observed sparks at the beginning of every evoked AP at numerous locations. Each spark was observed repeatedly at a fixed location and began during a limited interval after the AP peak. These sparks occurred with a probability of approximately unity. However, the chance that an LCC does not open during the interval when a spark is triggered is quite high (≈0.13). Therefore, because single channels open with a probability significantly lower than 1, more than one LCC must be available to ensure that sparks are triggered with a probability of approximately unity. We conclude that it is likely that a cluster of LCCs is involved in gating a cluster of ryanodine receptors at the beginning of an AP.

Abnormal myocyte Ca2+ homeostasis in rabbits with pacing-induced heart failure

To determine whether there are abnormalities in myocyte excitation-contraction coupling and intracellular Ca2+concentration ([Ca2+]i) homeostasis in pacing-induced heart failure (PF), we measured L-type Ca2+ current ( I Ca,L) and Na+/Ca2+exchanger current ( I Na/Ca) with voltage clamp and measured intracellular Na+ concentration ([Na+]i) and [Ca2+]iwith the use of sodium-binding benzofuran isophthalate (SBFI) and fluo 3 in ventricular myocytes isolated from control and paced rabbits. The peak systolic and diastolic levels and the amplitude of electrically stimulated [Ca2+]itransients (0.25 Hz, extracellular Ca2+ concentration = 1.08 mM) were significantly less in PF myocytes. Also, there was prolongation of the times to peak and decline of [Ca2+]itransients. I Ca,Ldensity was markedly decreased in PF myocytes. I Na/Ca at -40 mV elicited by rapid exposure to 0 Na+ solution with a rapid solution switcher was significantly reduced in PF myocytes, suggesting that the function of the Na+/Ca2+exchanger is impaired in these myocytes. In PF myocytes the decline of the [Ca2+]itransient when the Na+/Ca2+exchanger was abruptly disabled was markedly prolonged compared with the decline in control myocytes, consistent with depressed sarcoplasmic reticulum (SR) Ca2+-ATPase function. RNase protection assay showed decreased levels of Na+/Ca2+exchanger and SR Ca2+-ATPase mRNA in PF hearts, consistent with the function studies. We conclude that the functions of L-type Ca2+channels, Na+/Ca2+exchanger, and SR Ca2+-ATPase are impaired in myocytes from rabbit hearts with failure induced by rapid pacing. These abnormalities result in reduced [Ca2+]itransients and systolic and diastolic dysfunction and appear to account for the abnormal ventricular function observed.

The effect of exchanger inhibitory peptide (XIP) on sodium-calcium exchange current in guinea pig ventricular cells.

We investigated the effect of exchanger inhibitory peptide (XIP) on Na-Ca exchange current (INa-Ca) in guinea pig ventricular cells. Cells were voltage-clamped with microelectrodes containing 20 mM Na+ and 14.0 mM EGTA ([Ca]i = 100 nM). An outward putative exchange current was stimulated when extracellular Na+ was reduced from 144 mM to zero (Li+ replaced Na+). This outward current showed a significant dependence on extracellular Ca2+. When Na+ removal was delayed for up to 40 minutes (in the absence of extracellular K+ or the presence of 3.0 mM ouabain to block the Na+ pump), outward INa-Ca increased presumably because [Na]i increased. Time-dependent increases of outward current in the absence of K+ could be abolished by reapplication of K+, which presumably reactivates the Na+ pump and reduces intracellular Na+. This effect is blocked in the presence of 3.0 mM ouabain. The dependence of this current on extracellular Ca2+, its dependence on intracellular Na+, and activation by extracellular Na+ reduction, together with its resistance to ouabain all suggest that it is a Na-Ca exchange current. After dialyzing the cell with 10 microM XIP, outward INa-Ca was largely abolished. This indicates that XIP, which is a rather large molecule, can enter the heart cell via the microelectrode in sufficient quantities to inhibit exchange. Inward INa-Ca was blocked secondary to the blockade of outward INa-Ca. L-type Ca2+ current (ICa) was not measurably affected by XIP.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel Features of the Rabbit Transverse Tubular System Revealed by Quantitative Analysis of Three-Dimensional Reconstructions from Confocal Images

With scanning confocal microscopy we obtained three-dimensional (3D) reconstructions of the transverse tubular system (t-system) of rabbit ventricular cells. We accomplished this by labeling the t-system with dextran linked to fluorescein or, alternatively, wheat-germ agglutinin conjugated to an Alexa fluor dye. Image processing and visualization techniques allowed us to reconstruct the t-system in three dimensions. In a myocyte lying flat on a coverslip, t-tubules typically progressed from its upper and lower surfaces. 3D reconstructions of the t-tubules also suggested that some of them progressed from the sides of the cell. The analysis of single t-tubules revealed novel morphological features. The average diameter of single t-tubules from six cells was estimated to 448 +/- 172 nm (mean +/- SD, number of t-tubules 348, number of cross sections 5323). From reconstructions we were able to identify constrictions occurring every 1.87 +/- 1.09 microm along the principal axis of the tubule. The cross-sectional area of these constrictions was reduced to an average of 57.7 +/- 27.5% (number of constrictions 170) of the adjacent local maximal areas. Principal component analysis revealed flattening of t-tubular cross sections, confirming findings that we obtained from electron micrographs. Dextran- and wheat-germ agglutinin-associated signals were correlated in the t-system and are therefore equally good markers. The 3D structure of the t-system in rabbit ventricular myocytes seems to be less complex than that found in rat. Moreover, we found that t-tubules in rabbit have approximately twice the diameter of those in rat. We speculate that the constrictions (or regions between them) are sites of dyadic clefts and therefore can provide geometric markers for colocalizing dyadic proteins. In consideration of the resolution of the imaging system, we suggest that our methods permit us to obtain spatially resolved 3D reconstructions of the t-system in rabbit cells. We also propose that our methods allow us to characterize pathological defects of the t-system, e.g., its remodeling as a result of heart failure.