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Am J Physiol Heart Circ Physiol 287: H1906-H1912, 2004. First published July 8, 2004; doi:10.1152/ajpheart.00356.2004
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TRANSLATIONAL PHYSIOLOGY

Adenylyl cyclase type VI corrects cardiac sarcoplasmic reticulum calcium uptake defects in cardiomyopathy

Tong Tang,1,3 Mei Hua Gao,1,3 David M. Roth,2,3 Tracy Guo,1,3 and H. Kirk Hammond1,3

1Departments of Medicine and 2Anesthesiology, University of California San Diego, San Diego 92093; and 3Veterans Affairs San Diego Healthcare System, San Diego, California 92161

Submitted 13 April 2004 ; accepted in final form 30 June 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Calcium malfunction plays a central role in heart failure. Here, we provide evidence that adenylyl cyclase type VI restores sarco(endo)plasmic reticulum 2a (SERCA2a) affinity for calcium and maximum velocity of cardiac calcium uptake by sarcoplasmic reticulum in murine dilated cardiomyopathy. Restoration of normal SERCA2a affinity for calcium is associated not only with decreased phospholamban protein expression but also with increased phospholamban phosphorylation by PKA activation. The ratio of phosphorylated ryanodine receptor 2 (RyR2) to RyR2 protein was increased, but the amount of phosphorylated RyR2 was unaffected. These data provide a possible mechanism by which adenylyl cyclase type VI (in contrast to other signaling elements associated with increased cAMP generation) has a salutary effect in the failing heart.

cAMP; {beta}-adrenergic receptor; myocardium; heart failure

HEART FAILURE is associated with abnormalities in cardiac cAMP generation and calcium handling, two interdependent pathways that determine cardiac contractile function. However, efforts to restore intracellular cAMP levels to normal through pharmacological means have not been associated with successful clinical outcomes (3), and agents that directly influence calcium signaling have not yet been very successful. These two tightly linked dysfunctional pathways in heart failure (cAMP generation and calcium signaling) remain elusive targets in heart failure therapeutics.

Adenylyl cyclase (AC) type VI (ACVI), a dominant AC isoform in mammalian cardiac myocytes (39), appears to have favorable effects on heart function, although it is associated with increased cAMP generating capacity. For example, cardiac-directed expression of ACVI in murine cardiomyopathy increases cardiac function, attenuates myocardial hypertrophy, and increases survival (33, 34). However, when cardiac-directed {beta}-adrenergic receptor ({beta}AR) expression is used to treat this same model, the life span is shortened (11). Clearly there are marked differences in effects that are evoked by these two elements in the {beta}AR-Gs-AC signaling pathway, although both strategies increase cAMP. The objective of the current study was to determine whether there is a unique link between AC and calcium signaling that may explain the salutary effects of this pivotal effector molecule in heart failure.

Calcium plays a crucial role in controlling the cardiac contractile process (4, 31). During every heartbeat, calcium is taken up and then released from the sarcoplasmic reticulum (SR). The calcium pump responsible for cardiac SR calcium uptake is sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) (26). Failing hearts exhibit defective calcium uptake (13, 17, 22, 29, 35). Recent attention has been focused on phospholamban (PLN) expression and function within this context (8, 20).

Because of these observations (i.e., the potential importance of altered PLN expression and calcium signaling in heart function and the nonuniformity of responses between {beta}AR and ACVI gene expression in heart failure), we wondered whether ACVI was associated with specific effects on PLN expression and calcium signaling. To test this hypothesis, we investigated how cardiac-directed ACVI expression affects SR calcium uptake in cardiomyopathy. We demonstrated that ACVI expression is associated with increased SR calcium uptake, while having no effect on SR calcium release. This process is mediated by cAMP-dependent PKA and PLN phosphorylation and is associated with reduced PLN expression. Thus cardiac-directed expression of ACVI has salutary effects in the failing heart, at least in part, via mechanisms involving calcium uptake.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals. This study was approved by the Animal Use and Care Committee of the Veterans Affairs San Diego Healthcare System, in accordance with American Association for Accreditation of Laboratory Animal Care guidelines. ACVI mice (14) were crossbred with G{alpha}q transgenic mice (9, 33, 34), and the offspring were genotyped with the use of genomic DNA purified from tail clip as previously described (14). Left ventricles from 2.5-mo-old transgene negative, ACVI, G{alpha}q, and ACVI/G{alpha}q littermate mice were rinsed with PBS, snap-frozen in liquid nitrogen, and stored at –80°C.

Homogenization of left ventricle. Left ventricles were homogenized (4°C) in homogenization buffer (25 mM Tris·HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM {beta}-mercaptoethanol, 50 mM {beta}-glycerophosphate, 10 mM NaF, and 1 mM Na3VO4), except for the calcium uptake assay. Protease inhibitor cocktail (Roche; Indianapolis, IN) was included in the homogenization buffer.

PKA activity assay. Left ventricular homogenates were centrifuged at 14,000 g for 5 min at 4°C, and the supernatant was used for determination of intrinsic PKA activity using a cAMP-dependent PKA assay system (SignaTECT, Promega; Madison, WI).

cAMP phosphodiesterase activity assay. Left ventricular homogenates were extracted with 1% Triton X-100 (final concentration) on ice for 10 min with occasional vortexing. The resulting supernatant from 10,000 g centrifugation (5 min, 4°C) was used for measurement of phosphodiesterase (PDE) activities. Total cAMP PDE activity was measured following previously reported methods (43).

PKC activity assay. Left ventricular homogenates were separated into membrane and cytosolic fractions by centrifugation at 100,000 g for 30 min after previously reported methods (12). PKC activities were determined by measuring 32P incorporation of biotinylated-specific PKC substrate peptide in the absence and presence of calcium-diacylglyerol-phosphatidylserine with the use of components from SignaTECT PKC Assay System (Promega).

Calcium uptake. Frozen left ventricles were homogenized in buffer (10 mM imidazole, pH 7.0, 0.3 M sucrose, and 1 mM dithiothreitol, 10 mM NaF). Protease inhibitor cocktail (Roche) was included in the homogenization buffer. SR calcium uptake was measured by the modified Millipore filtration technique, and initial calcium uptake rates were calculated by linear regression (25).

Antibodies. Mouse monoclonal antibodies to SERCA2a and PLN were obtained from Affinity BioReagents (Golden, CO); rabbit antibody to phospho-Ser16-PLN was obtained from Upstate (Charlottesville, VA); and rabbit antibodies against ryanodine receptor type 2 (RyR2) and phospho-RyR2 (32) were provided by A. R. Marks (Columbia University). Rabbit antibodies to G{alpha}q and ACVI were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Western blot analysis. Left ventricular homogenates were analyzed by Western blotting to compare SERCA2a, PLN, phospho-PLN, RyR2, and phospho-RyR2 levels. Expression of cardiac ACVI and G{alpha}q was also confirmed by Western blot analysis. Briefly, left ventricular homogenates were denatured at 95°C for 3 min in Laemmli buffer. Samples were subjected to SDS-PAGE in polyacrylamide gels. After electrophoresis, proteins were electro-transferred to a polyvinylidene difluoride membrane (Bio-Rad; Hercules, CA). Equal loading of samples and even transfer efficiency were monitored with the use of 0.5% Ponceau S staining of the blot membrane. The blot membrane was then incubated in a blocking buffer (5% milk, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20) for 1 h and then incubated with the indicated antibodies overnight. Binding of the primary antibody was detected with the use of horseradish-conjugated secondary antibodies and enhanced chemiluminescence reagents (Amersham Biosciences; Piscataway, NJ). The blot membrane was then stripped by incubation in 0.3 M glycine (pH 2) for 2 h and used for additional protein expression detection. Quantification of protein expression was performed with the use of Gel-Pro analyzer (Media Cybernetics; Silver Spring, MD).

Statistical analysis. Results are shown as means ± SE. Differences among the four group means were detected with the use of one-way ANOVA. When the overall ANOVA indicated a rejection of the null hypothesis (P < 0.05), we used Bonferroni's t-test to determine whether there were differences between specific groups (two at a time). ANOVA and Bonferroni t-tests were performed with the use of Prism software (GraphPad; San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ACVI normalizes decreased calcium uptake in cardiomyopathy. To explore the mechanism by which ACVI improves global cardiac function in G{alpha}q-associated cardiomyopathy, we compared ATP-dependent initial SR calcium uptake rate in left ventricular homogenates from transgene negative, ACVI, G{alpha}q, and ACVI/G{alpha}q mouse hearts. Calcium uptake rates were impaired in cardiomyopathic hearts (Fig. 1). Analysis of these data indicated that not only SERCA2a affinity for calcium but also the maximum velocity (Vmax) of calcium uptake was decreased in the G{alpha}q mice (Fig. 1; Table 1). These abnormalities in calcium uptake were completely normalized by co-expression of ACVI in the cardiomyopathic background (Fig. 1 and Table 1). Overexpression of ACVI in the normal hearts did not have any effect on SERCA2a affinity for calcium and Vmax of calcium uptake. The ACVI-associated restoration of normal calcium uptake would be expected to increase cardiac contractility and relaxation as we have observed in these mice (33, 34).



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  		Fig. 1. A: adenylyl cyclase type VI (ACVI) expression in the G{alpha}q cardiomyopathic background increased maximum velocity (Vmax; G{alpha}q vs. ACVI/G{alpha}q; P < 0.05), whereas it had no effect on sarcoplamic reticulum (SR) calcium uptake in control mice. B: calcium uptake at different free calcium concentrations was normalized to Vmax and EC50 determined. ACVI normalized defective SR calcium uptake activity in cardiomyopathic hearts. Eight hearts from each group were used for calcium uptake analysis. Values are means ± SE. Vmax and EC50 were determined and data tested for differences among groups using one-way ANOVA, which showed that differences were present in Vmax (P = 0.012) and in EC50 (P < 0.006). Post hoc testing (Bonferroni t-test) was used to detect differences between pairs of group means. ACVI expression in the cardiomyopathic background decreased EC50 (G{alpha}q vs. ACVI/G{alpha}q; P < 0.05).

 

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  		Table 1. SERCA2a affinity for calcium

 
ACVI counteracts changes in PLN expression and phosphorylation at Ser16 in cardiomyopathy. To explore the importance of PLN in increased SERCA2a affinity for calcium conferred by ACVI expression in cardiomyopathy, we examined the extent of PLN protein expression and Ser16 phosphorylation by Western blotting analyses. PLN protein expression was increased in hearts from G{alpha}q mice (Fig. 2, A and B). Concurrent ACVI expression in this cardiomyopathic background was associated with reduced PLN protein expression (Fig. 2, A and B).



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  		Fig. 2. ACVI regulated phospholamban (PLN) expression and phosphorylation. A: representative Western blot analysis of PLN expression in transgene negative (Con), ACVI, G{alpha}q, and ACVI/G{alpha}q mouse hearts. The membrane was stripped and reprobed with an antibody against phospho-Ser16-PLN. B: concurrent cardiac ACVI expression in the G{alpha}q background was associated with reduced PLN expression. C: concurrent cardiac ACVI expression in the G{alpha}q background was associated with increased PLN phosphorylation. ANOVA indicated that a difference in group means was present. *P < 0.05, G{alpha}q vs. ACVI/G{alpha}q; n = 8 for each group. In B and C, bars represent mean values; error bars denote SE.

 
The level of PLN phosphorylation at the Ser16 site was reduced in G{alpha}q hearts (Fig. 2, A and C), despite increased PLN expression in hearts of these animals. ACVI increased PLN phosphorylation in the cardiomyopathic background but had no such effect in the absence of cardiomyopathy (Fig. 2, A and C). Thus ACVI increases SERCA2a affinity for calcium not only by reducing PLN protein expression but also by promoting PLN phosphorylation at Ser16 in cardiomyopathic hearts.

To explore the possibility that ACVI increased the Vmax of calcium uptake in G{alpha}q cardiomyopathic background by increasing SERCA2a expression, we assessed SERCA2a protein expression by Western blot analysis. ANOVA showed group differences in SERCA2a expression (P = 0.015); post hoc testing (Bonferroni t-test) showed that SERCA2a expression was decreased in G{alpha}q mice compared with transgene negative siblings (Control: 723 ± 59 densitometry units, n = 8; G{alpha}q: 514 ± 29 densitometry units, n = 8; P < 0.05), as was previously reported (38). We found no change in SERCA2a protein expression when ACVI was expressed in this cardiomyopathic background (G{alpha}q: 514 ± 29 densitometry units, n = 8; ACVI/G{alpha}q: 572 ± 29 densitometry units, n = 8; P > 0.05).

ACVI increases PKA activity in cardiomyopathy. To explore the mechanism by which ACVI increases PLN phosphorylation, we compared PKA activities in left ventricular homogenates from transgene negative, ACVI, G{alpha}q, and ACVI/G{alpha}q mice. PKA activity in hearts of ACVI mice was unchanged but was decreased in cardiomyopathic hearts (Fig. 3A). Co-expression of ACVI in this cardiomyopathic background increased PKA activity to normal levels (Fig. 3A). Changes in PLN phosphorylation reflected changes in PKA activities, indicating ACVI controls PLN phosphorylation by increasing PKA activity in cardiomyopathic hearts.



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  		Fig. 3. PKA, phosphodiesterase (PDE), and PKC activities in Con, ACVI, G{alpha}q, ACVI/G{alpha}q mouse hearts. A: concurrent cardiac ACVI expression in the G{alpha}q background was associated with increased PKA activity. ANOVA indicated that a difference in group means was present (P < 0.0002); *P < 0.01, G{alpha}q vs. ACVI/G{alpha}q; n = 8 in each group. B: PDE activity was reduced in hearts from G{alpha}q mice but unchanged by coexpression of ACVI. ANOVA indicated that a difference in group means was present (P < 0.001); *P < 0.01, G{alpha}q vs. Con and ACVI/G{alpha}q vs. Con; P > 0.05, G{alpha}q vs. ACVI/G{alpha}q; n = 8 in each group. C: calcium-diacylglycerol-phospholipid-dependent PKC activity in membrane fraction was reduced in hearts from G{alpha}q mice but unchanged by coexpression of ACVI. ANOVA indicated that a difference in group means was present (P < 0.0001); *P < 0.001, G{alpha}q vs. Con; and ACVI/G{alpha}q vs. Con; P > 0.05, G{alpha}q vs. ACVI/G{alpha}q; n = 8 in each group. Memb, membrane. D: calcium-diacylglycerol-phospholipid-dependent PKC activity in cytosolic fraction was reduced in hearts from G{alpha}q mice but unchanged by coexpression of ACVI. ANOVA indicated that a difference in group means was present (P < 0.0001); *P < 0.001, G{alpha}q vs. Con; and ACVI/G{alpha}q vs. Con; P > 0.05, G{alpha}q vs. ACVI/G{alpha}q; n = 8 in each group. Cyto, cytosolic. Bars represent mean values; error bars denote SE.

 
ACVI expression does not affect cardiac phosphodiesterase activity. To determine whether ACVI affects cardiac PDE activity, we measured total PDE activities in hearts from transgene negative, ACVI, G{alpha}q, and ACVI/G{alpha}q mice. Hearts of G{alpha}q mice showed decreased PDE activities. ACVI expression had no effect on PDE activity and did not increase PDE activity when expressed in the cardiomyopathic background (Fig. 3B).

ACVI expression does not affect cardiac PKC activity. To determine whether ACVI expression affects cardiac PKC activity, we measured calcium-diacylglyerol-phospholipid-dependent membrane and cytosolic PKC activities in transgene negative, ACVI, G{alpha}q, and ACVI/G{alpha}q mouse hearts. Hearts from G{alpha}q mice showed increased membrane and cytosolic PKC activities (Fig. 3, C and D), but ACVI expression did not influence these activities in cardiomyopathic or normal hearts (Fig. 3, C and D). The proportion of total cellular PKC activity that was found in the membrane fraction was similar in all four groups, ranging from 62% to 65%.

ACVI expression and cardiac RyR2 phosphorylation. To explore the possible role of ACVI on calcium release from SR, we studied RyR2 protein expression and phosphorylation. The RyR2 phosphorylation level was not altered by expression of ACVI or G{alpha}q alone (Fig. 4, A and B). However, RyR2 protein expression was increased in cardiomyopathic hearts, and decreased by co-expression of ACVI (Fig. 4, A and C). The ratio of phosphorlated RyR2 to RyR2 protein was increased (Fig. 4D) when ACVI was coexpressed in the cardiomyopathic background (ACVI/G{alpha}q vs. G{alpha}q) to a level that was statistically indistinguishable from Control mice (Fig. 4D).



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  		Fig. 4. ACVI effects on RyR2 expression and phosphorylation. A: representative Western blot analysis of phospho-RyR2 in transgene negative, ACVI, G{alpha}q, ACVI/G{alpha}q mouse hearts. The membrane was stripped and reprobed with an antibody against RyR2. B: concurrent cardiac ACVI expression in the G{alpha}q background did not affect RyR2 phosphorylation; n = 8 in each group. C: concurrent cardiac ACVI expression in the G{alpha}q background decreased RyR2 expression in hearts from G{alpha}q mice. ANOVA indicated that a difference in group means was present (P < 0.001). D: concurrent cardiac ACVI expression in the G{alpha}q background increased RyR2 phosphorylation to protein ratio. ANOVA indicated that a difference in group means was present (P < 0.002); *P < 0.01, G{alpha}q vs. ACVI/G{alpha}q; n = 8 in each group. Bars represent mean values; error bars denote SE.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The most important finding of this study was that ACVI restores SERCA2a affinity for calcium in murine dilated cardiomyopathy by regulating PLN inhibition of SERCA2a. These data provide a possible mechanism by which ACVI (in contrast to other signaling elements associated with increased cAMP generation) has a salutary effect in the failing heart.

Expression of G{alpha}q in cardiac myocytes is associated with reduced AC activity, impaired cardiac function, dilated cardiomyopathy, myocardial hypertrophy, and early mortality (9, 33, 34). In the present study, we showed that these cardiomyopathic hearts have reduced PKA activity, decreased PLN phosphorylation, and reduced SERCA2a affinity for calcium. Cardiac-directed ACVI expression increased AC and PKA activities, increased PLN phosphorylation, and fully corrected abnormal SERCA2a affinity for calcium in cardiomyopathic hearts. This effect was limited to the PKA signaling pathway: PKC activity and PDE activity were unaffected by AC expression. Thus ACVI specifically activated the PKA-PLN phosphorylation signaling pathway, which increased SERCA2a activity in cardiomyopathic hearts. These findings provide a potential mechanism by which ACVI improves cardiac contractility and relaxation in cardiomyopathy. These beneficial effects are not limited to G{alpha}q cardiomyopathy and do not reflect a peculiarity of transgenic models because we see favorable effects of exogenous ACVI gene transfer in pigs with pacing induced heart failure (23).

The objective of the present study was to determine whether there was a unique link between adenylyl cyclase and calcium signaling that may explain the salutary effects of ACVI in heart failure, an effect not shared by the other two members of this signaling pathway ({beta}AR and Gs). We show here that ACVI corrects calcium uptake defects in dilated cardiomyopathy. Cardiac-directed expression of {beta}1AR, in contrast, is associated with defective calcium handling, which precedes eventual cardiomyopathy (13). A distal element of the {beta}AR-Gs-AC signaling pathway, PKA, when expressed in hearts of transgenic mice, is also associated with defective calcium handling (1). Therefore, the beneficial effect of ACVI on calcium signaling is unique among the elements of the {beta}AR-Gs-AC signaling pathway.

Decreased generation of cAMP by cardiomyocytes is a hallmark of heart failure (6, 7, 10, 28, 33, 34). Decreased phosphorylation of PLN by cAMP-dependent PKA, which has been shown to release the inhibition of SR calcium uptake activity by unphosphorylated PLN, is also found in clinical heart failure (35–37). Expression of a PKA-pseudophosphorylated mutant of PLN increases SR calcium uptake, improves global cardiac function, and attenuates heart failure progression in an animal model of cardiomyopathy (18). Restoration of PLN phosphorylation and SR calcium uptake by ACVI seems to be the underlying mechanism for its improved calcium signaling. Interestingly, the major SR calcium release channel RyR2, also a substrate for PKA, is hyperphosphorylated in clinical heart failure (27). Hyperphosphorylation of RyR2 is associated with calcium leak from SR and PKA appears to be an important mediator for this process. Overexpression of an active form of PKA increases phosphorylation of both RyR2 and PLN (1). The effect of increased RyR2 phosphorylation on SR calcium release seems to override the effect of increased PLN phosphorylation on SR calcium uptake, resulting in myocardial hypertrophy, arrhythmia and sudden death (1). However, ACVI increases PLN phosphorylation but has no effect on RyR2 phosphorylation when expressed in the G{alpha}q cardiomyopathic background. However, the ratio of phosphorylated RyR2 to RyR2 protein was increased in cardiac samples from ACVI/G{alpha}q versus G{alpha}q mice. This ratio was statistically indistinguishable from control mice, and therefore, does not represent a hyperphosphorylated state. This difference may explain why PKA transgenic mice develop heart failure but ACVI transgenic mice do not (1, 14). The apparent specificity of ACVI-increased PLN phosphorylation warrants further study.

Another factor that distinguishes ACVI and PKA overexpression is the nature of PKA activation that results. ACVI does not increase basal cardiac AC activity (14, 15), and, as we have shown here, does not increase PKA activity in normal hearts. In contrast, PKA overexpression evokes sustained activation of PKA (1), a feature shared by many manipulations ({beta}AR overexpression, {beta}AR agonist stimulation, G{alpha}s overexpression, and PDE inhibition) that ultimately are deleterious to the heart (24). The absence of sustained PKA activation associated with ACVI overexpression may be an important component of its beneficial effects. We found decreased PKA activity in G{alpha}q hearts, which is different from a previous report (12). The nature for this discrepancy is unknown, but reduced PKA activity is consistent with reduced AC activity, which is associated with this model (11, 33, 34).

In addition to increased SERCA2a affinity for calcium in the hearts of ACVI/G{alpha}q mice compared with those from G{alpha}q mice, the Vmax of SR calcium uptake is increased (Fig. 1A; Ref. 42), a phenomenon seen also when SERCA2a is overexpressed in the heart (2). However, we found that SERCA2a protein levels were not altered in the hearts of ACVI/G{alpha}q mice compared with those from G{alpha}q mice, although SERCA2a expression is lower in G{alpha}q mice compared with nontransgenic mice, confirming previous work (38). PLN phosphorylation and expression might change SERCA2a affinity for calcium but not Vmax (21, 30). It is unlikely that the change of RyR2 phosphorylation to protein ratio or the change in calcium leak contribute to increased Vmax of calcium uptake since ruthenium red was included in the calcium uptake assay to inhibit maximally the SR calcium release channel RyR2. The precise molecular mechanism for altered Vmax will require further studies. Cardiac PKC activity may be linked to SERCA2a activity in heart failure (5, 16). We found that PKC activity was increased in G{alpha}q hearts, but improved calcium handling associated with concomitant ACVI expression was not associated with reduced PKC activity, suggesting that the mechanism was not related to PKC.

We have shown that PLN protein was increased in cardiomyopathic hearts, but PLN phosphorylation was decreased. ACVI expression increased PLN phosphorylation but decreased PLN protein expression. We speculate that phosphorylation of PLN facilitates PLN degradation through increased ubiquitination (40, 41). Reduced PLN expression associated with ACVI expression in cardiomyopathy provides an additional means for normalization of calcium uptake defects and blocking the development of heart failure.

Why should PLN ablation, which did not improve cardiac function in G{alpha}q cardiomyopathy (38), be different than reduction of PLN via ACVI expression, which did? Reduction in PLN that we observed was not via ablation of PLN, but was an indirect effect of ACVI expression. ACVI has effects on the cAMP-generating capacity that may increase LV function, in addition to its effects on PLN (14, 33, 34). It may be that reduced cardiac PLN and increased cAMP generating capacity are each (individually) insufficient to improve outcomes in heart failure. We speculate that the combination of these two methods, which appears to result from cardiac-directed ACVI expression, may be sufficient. Additional studies will be required to provide an unambiguous explanation that definitively links correction of impaired calcium handling and improvement of cardiac function in heart failure (19).

In conclusion, we have shown that ACVI restores SERCA2a affinity for calcium in murine dilated cardiomyopathy by regulating PLN inhibition of SERCA2a. These data provide a possible mechanism by which ACVI, compared with other signaling elements associated with increased cAMP generation, has a salutary effect in the failing heart.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grant 1-P01-HL-669411 (to H. K. Hammond) and Department of Veterans Affairs Merit Review Awards (to D. M. Roth and H. K. Hammond).


   ACKNOWLEDGMENTS
 
We thank Drs. W. Y. W. Lew, W. R. Giles, J. M. Harrer, and W. Zhang for helpful discussions, A. R. Marks for providing RyR2 and phospho-RyR2 antibodies, and G. W. Dorn II for providing the initial G{alpha}q transgenic mouse line from which we generated the four lines used in this study by cross-breeding with our ACVI line.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Kirk Hammond, 111A, VA San Diego Healthcare System, 3350 La Jolla Village Dr., San Diego, CA 92161 (E-mail: khammond{at}ucsd.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
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 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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