Response to Mattiazzi et al:
Our recent article in Circulation Research1 has received attention from a number of investigators in the field. Our goal in this research was to explore the hypothesis proposed by Marks and colleagues that PKA-mediated phosphorylation of the ryanodine receptor at Ser2808/09 is a central component of the sympathetic nervous system (SNS) regulation of cardiac contractility.2 Our experiments were mainly designed to address this issue. We used a genetically modified mouse in which phosphorylation of Ser2808/09 was eliminated by an alanine substitution (S2809/09A). Our results strongly supported the idea that PKA-mediated phosphorylation of Ser2808/09 has no significant functional role in SNS regulation of cardiac function.
In this issue of Circulation Research, Mattiazzi et al3 have written to the editor regarding our recent report.1 The purpose of our letter here is to respond to the 3 points made by Mattiazzi et al.3 First, we all agree that there is significant evidence that phosphorylation of Ser2808/09, either by PKA,2,4 or by CaMKII,5,6 has little functional significance in the response of the heart to sympathetic stimulation. As we reviewed in our recent report,1 many laboratories are in agreement on this issue, including Mattiazzi and colleagues,6 and this was the major conclusion of our report.
Mattiazzi et al3 point out that there appear to be other PKA phosphorylation sites on RyR2 and that these may have functional significance. Our recent study did not evaluate this possibility; however, Valdivia and colleagues, coauthors in the recent by MacDonnell et al,1 have been actively involved in this debate.4 We agree with Mattiazzi et al3 that there is substantial evidence that RyR2 can be PKA-phosphorylated at sites other than Ser2808/09, specifically Ser2030. Still, it is important to note that the issue of the contribution of phosphorylation-mediated alterations in RyR2 opening probability to SNS-mediated increases in cardiac contractility is yet to be fully resolved. In our recent report,1 we discussed the published studies that led us to conclude that SNS-mediated increases in contractility primarily involve increases in Ca2+ influx through the L-type Ca2+ channel and increased Ca2+ uptake and release by the sarcoplasmic reticulum. In our view, the most convincing data arguing against a substantial role for PKA-mediated alterations in RyR2 properties in SNS regulation of cardiac contractility are those that show that PKA has no effect on Ca2+ sparks in phospholamban deficient7 or S2808/09A mouse myocytes4 Clearly, there is still work that needs to be done to define if and how modulation of RyR2 properties contributes to regulation of cardiac contractility by the SNS.
The last point made by Mattiazzi3 relates to the idea that CaMKII can regulate RyR2 channel activity. Although there is no consensus as to what RyR2 regulation by CaMKII accomplishes (inhibition or activation of channel activity), there is strong evidence indicating that regulation occurs and we reviewed some of these data in our recent study.1 Mattiazzi et al3 appear to be have misinterpreted some of our statements. We did not state, nor do we believe, that increases in heart rate are the only mechanism for activation of CaMKII. In our in vivo experiments, we found that isoproterenol increased contractility in wild-type and S2808A mice to an equal extent and also increased heart rate. We were concerned that the increase in heart rate might have obscured our ability to see differences in contractility in S2808A mice, possibly by increasing the activity of CaMKII. Therefore, we performed a series of studies in rate-controlled isolated hearts. These studies again showed no differences in isoproterenol effects on cardiac contractility between wild-type and S2808A mice. We fully recognize and agree with Mattiazzi et al3 that CaMKII will be activated in both of the conditions we used. We did not wish to give the impression that CaMKII was not activated in rate-controlled hearts exposed to isoproterenol.
It is gratifying to see that the regulation of cardiac contractility still evokes such passionate research after all these years. There is still much to be learned, and novel model systems needed to explore the functional effects of PKA- and CaMKII-mediated phosphorylation of RyR2 are available for all to use.
Sources of Funding
MacDonnell S, García-Rivas G, Kubo H, Chen X, Scherman JA, Valdivia H, Houser SR. Adrenergic regulation of cardiac contractility does not involve phosphorylation of the cardiac ryanodine receptor at serine 2808. Circ Res. 2008; 102: e65–e72.
Mattiazzi A, Vittone L, Mundiña-Weilenmann C. Ca2+-calmodulin–dependent protein kinase phosphorylation of ryanodine receptor may contribute to the β-adrenergic regulation of myocardial contractility independently of increases in heart rate. Circ Res. 2008; 103.
Benkusky NA, Weber CS, Scherman JA, Farrell EF, Hacker TA, Powers PA, Valdivia HH. Intact β-adrenergic response and unaltered progression towards heart failure in mice with genetic ablation of a major PKA phosphorylation site in the cardiac ryanodine receptor. Circ Res. 2007; 101: 819–829.
Witcher DR, Kovacs RJ, Schulman H, Cefali DC, Jones LR. Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity. J Biol Chem. 1991; 266: 11144–11152.
Ferrero P, Said M, Sánchez G, Vittone L, Valverde C, Donoso P, Mattiazzi A, Mundiña-Weilenmann C. Ca2+/calmodulin kinase II increases ryanodine binding and Ca2+-induced sarcoplasmic reticulum Ca2+ release kinetics during beta-adrenergic stimulation. J Mol Cell Cardiol. 2007; 43: 281–291.
Guo T, Zhang T, Mestril R, Bers DM. Ca2+/calmodulin-dependent protein kinase II phosphorylation of ryanodine receptor does affect calcium sparks in mouse ventricular myocytes. Circ Res. 2006; 99: 398–406.