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Circulation Research. 2006;98:165-168
doi: 10.1161/01.RES.0000204551.09930.84
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(Circulation Research. 2006;98:165.)
© 2006 American Heart Association, Inc.

Editorials

"Ryanogate"

Who Leaked the Calcium?

Sheldon E. Litwin

From the Division of Cardiology, the University of Utah and the Salt Lake City Veterans Affairs Medical Center, Salt Lake City.

Correspondence to Sheldon E. Litwin, MD, The University of Utah, Cardiology Division, 4A100 SOM, 30 N 1900 E, Salt Lake City, UT 84132. E-mail Sheldon.Litwin{at}hsc.utah.edu



See related article, pages 235–244


Key Words: CaMKII • calcium • sarcoplasmic reticulum • heart failure • action potential • ryanodine

The sarcoplasmic reticulum (SR) is the major intracellular calcium storage depot in cardiac muscle. Cycling of calcium between the lumen of the SR and the myoplasmic space occurs repetitively during each heart beat. Excitation–contraction coupling in the heart begins when calcium entry through voltage-gated L-type calcium channels in the sarcolemma induces the opening of calcium release channels (also known as ryanodine receptors, or RyR) in the adjacent SR. The majority of the calcium that enters the cytosol during the early portion of each cycle is then resequestered into the SR lumen via the actions of the calcium uptake protein, (sarco)endoplasmic reticulum calcium adenosine triphosphatase (SERCA). Tight regulation of the timing and quantity of these intracellular calcium fluxes is critical to achieve graded contractility and relaxation of the heart muscle. Such control allows optimal matching of cardiac function to heart rate and metabolic needs of the body. The mechanisms of excitation–contraction coupling have been reviewed more thoroughly elsewhere.1

There has been a great deal of interest in the possibility that altered calcium homeostasis in the cardiac myocyte is the fundamental abnormality in the failing heart.2 The most widely accepted hypothesis to explain myocyte dysfunction is that reduced abundance or activity of SERCA protein directly results in a lower rate of calcium uptake by the SR. In this scenario, reduced SR uptake results in lower SR calcium content, which then produces lower SR calcium release and reduced contractility. However, the "SERCA-centric" view of heart failure is at best only a partial explanation of the cellular abnormalities that occur in failing hearts.3–5 We now recognize that many other highly complex mechanisms in addition to SERCA expression regulate SR calcium uptake and release. In particular, posttranslational modifications of SR proteins are known to be extremely important components in the regulation of calcium movements.

In the early 1980’s, Lakatta and colleagues observed that quiescent cardiac muscle had spontaneous fluctuations in the intensity of scattered laser light which they proposed represented spontaneous calcium release from the SR.6,7 More than a decade later, Cheng et al visualized elementary calcium release events in cardiac myocytes with the aid of a confocal microscope.8 These events, termed "calcium sparks," represent the calcium release from a single cluster of ryanodine receptors, or a calcium release unit. Normally, the coordinated occurrence of many sparks during the early portion of the action potential summates to produce a rapidly peaking intracellular calcium transient. Termination of spark release precedes the decline in bulk cytosolic calcium. In 2000, we reported that myocytes from infarcted rabbit hearts had a dyssynchronous and prolonged pattern of calcium spark production that potentially contributed to a lower peak, and slower upstroke and down stroke of the calcium transient.9 Subsequently, others have seen similar phenomena in both animal and human myocytes.10–15 The same year, Marks’ group made the discovery that the RyR was part of a large macromolecular complex that contained several accessory proteins, including protein kinase A (PKA), FKBP12.6, and protein phosphatases (PP1 and PP2A).16 In their seminal report, they proposed that in failing myocardium "hyperphosphorylation" of the RyR caused it to have a higher sensitivity to calcium, and thus to become functionally "leaky." The net effect of the SR calcium leak was thought to be a reduction in SR calcium content. Although substantial data have accumulated that support the general hypothesis put forth by these investigators, there has been some controversy regarding the specific sites of RyR phosphorylation, the role of FKBP12.6 dissociation, the kinase responsible for the phosphorylation, and the functional effects of such phosphorylation.17–19

Several of the key proteins that are involved in excitation–contraction coupling in the heart are regulated by phosphorylation at serine or threonine residues. In the normal heart, increased phosphorylation occurs during periods of increased sympathetic stimulation, such as during exercise. Increased phosphorylation tends to increase the amplitude and rate of cytosolic calcium fluxes with a net effect of increasing contractility and relaxation rates. At any point in time, the phosphorylation state of these proteins reflects the balance of local kinase and phosphatase activity. PKA is the major enzyme responsible for phosphorylation of calcium cycling proteins in the heart during beta adrenergic signaling. In recent years, a second kinase that is activated by calcium-calmodulin has been the subject of significant interest.20,21 Calcium/calmodulin-dependent kinase (CaMK) is a ubiquitous protein that is sensitive to the amplitude and rate of calcium spikes.22 In neurons, CaMK is thought to be responsible for long-term potentiation.21 In the heart, a specific isoform designated CaMKII{delta} mediates facilitation of L-type calcium currents.23,24 CaMKII expression appears to be increased in cardiac hypertrophy and heart failure,25 and enhanced CaMKII activity has been implicated as a cause of arrhythmias when action potential duration is prolonged.20,26 Recently, selective overexpression of CaMKII in the mouse heart was found to cause cardiomyopathy and premature death.25 Conversely, genetically modified mice that express a CaMKII inhibitor in the heart are more resistant to pathological remodeling and dysfunction after myocardial infarction or excessive beta adrenergic stimulation.27 Thus, substantial evidence suggests that this kinase is important in both normal and pathological cardiac function.

In this issue of Circulation Research, Kohlhaas et al add to the growing body of data showing that CaMKII plays a key role in the regulation of cardiac excitation–contraction coupling.28 In this article, the investigators used adenoviral-mediated overexpression of the cytosolic splice variant of CaMKII{delta} in short-term (24 hours) cultures of adult rabbit cardiac myocytes. The major findings were that CaMKII overexpression caused: (1) increased "leak" of calcium from the SR manifest as an increased frequency of resting calcium sparks normalized to SR calcium content; (2) markedly reduced SR calcium content; and (3) surprisingly normal calcium transients and cellular contractions. These effects occurred in the absence of any major changes in calcium handling protein expression (SERCA, phospholamban, Na+–Ca2+ exchanger) and appear to be mediated in large part by phosphorylation of ryanodine receptors. The RyRs showed increased phosphorylation at both Ser2809 (the putative PKA site) and Ser2815 (the CaMKII site).29 The findings expand on earlier work from this group by now showing that acute CaMKII overexpression causes SR calcium leak even in the absence of adaptive changes in other proteins and in the absence of a generalized "heart failure milieu." The similar findings in rabbit myocytes in the present study, as compared with mouse myocytes in the prior study, suggest that the phenomenon of CaMKII-induced SR leak is not species specific.

An intriguing aspect of this study is that the cytosolic calcium transients and cellular contractions were remarkably normal in myocytes with increased CaMKII expression, despite a markedly reduced SR load. Presumably, this occurred because the low SR content was precisely counterbalanced by an increase in RyR sensitivity and an increase in the amplitude of the L-type calcium current. The tendency of myocytes to quickly adapt to perturbations in RyR sensitivity and return to steady state amplitude of calcium transients has been well documented.30 The same seems to be true in the setting of acute CaMKII expression where the negative inotropic (SR unloading) and positive inotropic (enhanced RyR sensitivity) effects appear to be balanced (Figure 1A and 1B). However, under normal physiological conditions, enhanced CaMKII activity is an important mechanism for increasing contractility, particularly during increased heart rate.29 Because CaMKII is sensitive to the frequency of calcium oscillations,22 it is ideally suited to function as part of the normal frequency-dependent increase in cardiac force production. Increasing the open probability of the RyR, enhancing SR calcium uptake, and facilitation of ICa are all likely contributors to the positive force-frequency relationship in normal myocardium (Figure 1C).29


Figure 1
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  		Figure 1. Hypothetical model showing the relationships between sarcolemmal calcium fluxes, sarcoplasmic reticulum (SR) calcium load, SR fractional release, SR reuptake, and the resulting cytosolic calcium transient. A, Under normal conditions, sarcolemmal calcium influx (L-type calcium current and reverse sodium calcium exchange) is balanced with calcium efflux (forward sodium-calcium exchange and the calcium pump). Normal SR calcium load is shown in light blue, and the portion of released calcium is shown in the dotted circle. B, Under conditions of acute CaMKII overexpression SR content is reduced because of increased calcium leak. However, overall release is maintained because L-type calcium current is increased and fractional SR release in increased. The net calcium transient is unaltered. C, In the normal myocyte, increased rate activates CaMKII. This facilitates (increases) calcium current, increases fractional SR release, and accelerates calcium uptake. The net effect is an increased calcium transient with a faster rate of fall. D, In heart failure, the combination of increased CaMKII activity, action potential prolongation, and potentially numerous other factors, results in SR leak, reduced SR content, poorly synchronized SR release. The net effect is reduced amplitude and slowed kinetics of the calcium transient.

If CaMKII activation is a normal means for increasing contractility during increased heart rate and contractility is preserved with acute CaMKII overexpression (Figure 1C), why would increased CaMKII activity cause or contribute to heart failure? It appears that increased CaMKII activity alone is insufficient to account for the heart failure phenotype. One possible explanation for this quandary is that other changes in the myocyte tend to accentuate either the beneficial or detrimental affects of CaMKII. In other words, under the right conditions enhanced CaMKII activity can either enhance or decrease contractility (Figure 2). I propose the hypothesis that changes in action potential morphology are a modifying factor that interact with increased CaMKII activity to produce either the normal force–frequency relationship or the heart failure phenotype. During periods of increased heart rate, a key adaptation is shortening of action potential duration. Action potential shortening not only has the effect of allowing the heart to completely contract and relax during shorter cardiac cycles, but it may help to synchronize calcium release from the SR. This occurs because the driving force for calcium entry through open L-type calcium channels is increased when repolarization is accelerated and the channels also close or inactivate more quickly.31 Thus, in the setting of increased heart rate, increased RyR open probability does not produce ill effects because spark production is well synchronized and controlled. Conversely, action potential prolongation and attenuation of the early rapid repolarization phase (phase 1) tend to desynchronize SR release.11,12 Some data suggest that CaMKII is specifically activated at membrane potentials corresponding to the plateau of the cardiac action potential.32 There is broad acknowledgement that action potential duration is increased in most forms of cardiac hypertrophy and failure. Therefore, it seems plausible that the combination of enhanced CaMKII activity and action potential remodeling in a variety of heart diseases could collectively conspire to produce microscopic heterogeneity in calcium release. It is easy to envision how loss of both temporal synchronization and spatial homogeneity of calcium sparks could rob from the efficiency of the usual carefully orchestrated calcium fluxes in the cardiac myocyte (Figure 1D).


Figure 2
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  		Figure 2. Proposed model in which opposing changes in action potential morphology and duration modify the effects of CaMKII activation. In normal myocytes, increased heart leads to increased CaMKII activation and increased RyR open probability. However, action potential shortening maintains spark synchrony and hence contractility increases. In hypertrophy or failure, CaMKII activation also increases RyR open probability. However, action potential prolongation allows sparks to become unsynchronized and hence contractility is impaired.

In support of the above ideas and the concept that CaMKII is an important mediator of altered calcium release in heart failure, we previously reported that increased stimulation rate of myocytes from diseased hearts worsened the dyssynchrony of calcium sparks.9 Enhanced rate would be expected to further increase CaMKII activation and to exacerbate the underlying pathophysiology if CaMKII were involved. Secondly, we found that beta adrenergic stimulation improved the synchronization of the sparks in myocytes from infarcted hearts. If PKA-mediated RyR phosphorylation were the major cause of the impaired calcium dynamics, we would have expected the opposite result. However, if beta adrenergic stimulation shortened action potential duration and enhanced SR calcium content, then it might partially overcome unsynchronized calcium release caused by CaMKII activation.

In summary, the current work supports the notion that CaMKII-mediated RyR phosphorylation leads to increased sensitivity of these channels. It appears that this increases SR "leak" which leads to unloading of the SR. These findings challenge the notion that impaired SR calcium uptake is the major abnormality in failing myocardium. They also raise additional questions about the relative importance of PKA and CaMKII activity in heart failure. Despite the potentially deleterious effects of CaMKII activation, in otherwise normal myocytes, CaMKII-mediated SR unloading is counterbalanced by an increase in SR fractional release. Although CaMKII seems like an attractive therapeutic target in heart failure, we should pause to recall that CaMKII contributes importantly to the normal cardiac response to increased heart rate (enhanced contractility and faster relaxation) and it is a critical enzyme in multiple organs and tissues throughout the body. The dual and seemingly antithetical roles of CaMKII to decrease SR content but increase fractional release certainly will be the focus of investigations for years to come.


*    Acknowledgments
 
Dr Litwin is supported by grants from the Department of Veterans Affairs and the National Institutes of Health (HL70525)


*    Footnotes
 
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.


*    References
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Related Article:

Increased Sarcoplasmic Reticulum Calcium Leak but Unaltered Contractility by Acute CaMKII Overexpression in Isolated Rabbit Cardiac Myocytes
Michael Kohlhaas, Tong Zhang, Tim Seidler, Darya Zibrova, Nataliya Dybkova, Astrid Steen, Stefan Wagner, Lu Chen, Joan Heller Brown, Donald M. Bers, and Lars S. Maier
Circ. Res. 2006 98: 235-244. [Abstract] [Full Text] [PDF]



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