Adenylyl Cyclase Subtype–Specific CompartmentalizationNovelty and Significance
Differential Regulation of L-Type Ca2+ Current in Ventricular Myocytes
Rationale: Adenylyl cyclase (AC) represents one of the principal molecules in the β-adrenergic receptor signaling pathway, responsible for the conversion of ATP to the second messenger, cAMP. AC types 5 (ACV) and 6 (ACVI) are the 2 main isoforms in the heart. Although highly homologous in sequence, these 2 proteins play different roles during the development of heart failure. Caveolin-3 is a scaffolding protein, integrating many intracellular signaling molecules in specialized areas called caveolae. In cardiomyocytes, caveolin is located predominantly along invaginations of the cell membrane known as t-tubules.
Objective: We take advantage of ACV and ACVI knockout mouse models to test the hypothesis that there is distinct compartmentalization of these isoforms in ventricular myocytes.
Methods and Results: We demonstrate that ACV and ACVI isoforms exhibit distinct subcellular localization. The ACVI isoform is localized in the plasma membrane outside the t-tubular region and is responsible for β1-adrenergic receptor signaling–mediated enhancement of the L-type Ca2+ current (ICa,L) in ventricular myocytes. In contrast, the ACV isoform is localized mainly in the t-tubular region where its influence on ICa,L is restricted by phosphodiesterase. We further demonstrate that the interaction between caveolin-3 with ACV and phosphodiesterase is responsible for the compartmentalization of ACV signaling.
Conclusions: Our results provide new insights into the compartmentalization of the 2 AC isoforms in the regulation of ICa,L in ventricular myocytes. Because caveolae are found in most mammalian cells, the mechanism of β- adrenergic receptor and AC compartmentalization may also be important for β-adrenergic receptor signaling in other cell types.
- adenylyl cyclase type 5
- adenylyl cyclase type 6
- L-type Ca2+ current
- ventricular myocytes
- calcium channel
- adrenergic receptor
Adenylyl cyclase (AC)1,2 represents one of the principal effector molecules in the β-adrenergic receptor (βAR) signaling pathway, responsible for the conversion of ATP to the second messenger, cAMP. cAMP activates protein kinase A, which phosphorylates several key Ca2+-cycling proteins in cardiac myocytes, including phospholamban, ryanodine receptors, troponin I, and L-type Ca2+ channels (Cav1.2). Phosphorylation of these target proteins increases cytoplasmic Ca2+, resulting in an increased contractile response of cardiomyocytes. Unlike other components of the βAR signaling pathway, overexpression of AC does not promote cardiac hypertrophy, making it an attractive therapeutic target for heart failure.3
The mammalian AC gene family contains at least 10 members4 with several AC isoforms detected in the heart at the transcript level; however, only 2 isoforms (ACV and ACVI) are found in abundance.5,6 These 2 isoforms share ≈65% protein sequence identity, and both belong to a group of Ca2+-inhibitable ACs, which can be inhibited by physiological concentrations of Ca2+.5,7 A fundamental difference between the 2 isoforms is that ACV seems to cause detrimental effects in the aging model of cardiomyopathy, whereas overexpression of the ACVI isoform confers beneficial effects in models of cardiac hypertrophy.8,9
Subcellular compartmentalization of the βAR signaling proteins allows a variety of biological functions to be regulated at the same time by only a few βAR subtypes.10,11 Signaling proteins that are located close to each other can form a working unit, also called a signalosome. Different subcellular localizations have been shown to occur through a number of scaffolding proteins. Previous studies by Insel etal12 and Ostrom et al13 have provided insights into the roles of the caveolin-rich domain in subcellular localization of AC and βARs in cardiac myocytes. In addition, it has been documented that the interactions among different compartments of the βAR signaling are limited by phosphodiesterase (PDE)-dependent cAMP degradation.14–16
Caveolin is an essential molecule for the formation of caveolae, or membrane invaginations, which are found in most cell types.17 Caveolae increase the cell surface area, which is important for vesicular trafficking, signal transduction, and macromolecular transport. In the heart, the most abundant isoform of caveolin is caveolin-3, which is associated with long membrane invaginations known as transverse tubules (t-tubules) in addition to the surface membrane.18
Compartmentalization contributes to the complexity of subcellular organization, and alterations in the signaling pathway of a compartment could lead or contribute to cardiac pathology. For example, an increase in β2AR reactivity or redistribution of β2AR from t-tubules could contribute to cardiac toxicity during the progression of cardiomyopathy.19 Although the concepts of compartmentalization are well documented, there is still limited understanding of how different compartments are organized and of some of the key effectors in these specialized regions. Specifically, it is not known whether there is similar subcellular compartmentalization of the key effector molecules, ACV and ACVI, in the βAR signaling pathways. Moreover, it has not been established whether different βAR subtypes, namely β1AR and β2AR, mediate their downstream effects via different isoforms of ACs in the heart. Indeed, new insights into the distinct localization and function of the 2 main AC isoforms will help to shed light on these critical effector molecules during the development and progression of cardiac hypertrophy and failure, possibly providing new therapeutic rationales for these common cardiac conditions.
To accomplish these goals, we took advantage of ACV, ACVI, and ACV/ACVI knockout models to test the central hypothesis that there is subcellular compartmentalization of the 2 main AC isoforms in ventricular myocytes. ICa,L was recorded as a robust readout of the βAR signaling. The effects of β1AR versus β2AR signaling via the 2 AC isoforms were tested. Moreover, we provide new mechanistic insights into the pivotal roles of caveolin-3 as a scaffolding protein, which links the specific isoform of AC and PDE, leading to the specialized subcellular organization and forming a separate functional compartment. Finally, we provide evidence that caveolin-3 represents a critical molecule for the compartmentalization of the βAR signaling cascade in cardiac myocytes.
Detailed Materials and Methods are provided in the Online Data Supplement.
All animal care and use procedures were approved by the University of California, Davis Institutional Animal Care and Use Committee. Experiments were performed in accordance with National Institutes of Health and institutional guidelines. Genetically targeted mouse models, including ACV knockout (ACV KO)20 and ACVI knockout (ACVI KO),7 were used for the studies. The double-knockout (ACV/ACVI KO) mice were generated by crossing the homozygous single KO mice.
Single left ventricular myocytes were isolated from 10- to 12-week-old animals. All experiments were performed using the conventional whole-cell patch-clamp technique at room temperature. In some experiments, a detubulation technique consisting of an osmotic shock to internalize the t-tubules was used.21
Western Blot Analyses
Analyses were performed as previously described.22 Primary antibodies used in the study include anti–β-tubulin (Abcam ab6046, 1:4000 dilution) and anti–Cav1.2 (Sigma C1603, 1:200 dilution) antibodies.
The Rosetta-Membrane de novo method23 was used for structure prediction of caveolin. We first generated 10 000 caveolin models of the transmembrane regions of caveolin followed by model clustering as described previously.23 The center model of the largest cluster was then used for modeling of N and C termini of caveolin using the Rosetta loop modeling method.24
The ACV and mutated ACV molecule were modeled using the Protein Homolog/analogy Recognition Engine V1.0 (PHYRE) database.25 Only the peptide segment before the first transmembrane segment was included in the modeling. After both ACV models were created, the peptides were truncated to 20 amino acids to mimic our experiments (amino acids 84–104 of the mouse ACV sequence).
Molecular graphics and analyses were performed using the University of California, San Francisco Chimera package.26 Dock 6.4 was used for the docking of the peptides.27,28 The docking of the peptides used a rigid algorithm (the individual parts of the peptide were kept in the same orientation). The caveolin-3 peptide was treated as a receptor, and the ACV wild-type and mutant peptide was treated as aligand. The model was run 6 times with a range of grid from 0.1 to 10 Å to confirm the results.
ACVI Isoform Is Critical for the Observed βAR Enhancement of ICa,L in Mouse Ventricular Myocytes
We first evaluated the distinct role of ACV and ACVI in βAR stimulation of ICa,L using the whole-cell patch-clamp technique (Figure 1). Ventricular myocytes were isolated from 4 different groups of animals, including wild type (WT), ACV KO, ACVI KO, and the double KO (ACV/ACVI KO). Application of isoproterenol (ISO) at a concentration of 1 µmol/L significantly increased ICa,L in WT ventricular myocytes (Figure 1A). Similar results were observed in the ACV KO group (Figure 1A, center). Remarkably, ISO had no effects on ICa,L in myocytes isolated from ACVI KO (Figure 1A, bottom) or ACV/ACVI KO myocytes (Online Figure I). In addition, the basal current was significantly decreased (−1.6±0.4 pA/pF at 0 mV) in ACVI KO myocytes (Figure 1A, bottom). There was no statistical difference in ICa,L between ACVI KO and the double-KO myocytes. Summary data for the current density elicited at 0 mV are shown in Figure 1B.
ICa,L was recorded 10 minutes after patch rupture for the current to stabilize. Online Figure IIA shows the time course of the current at baseline and 30 minutes after ISO, demonstrating a relatively stable current in our recording conditions. Indeed, in all our patch-clamp experiments, we have ensured that current recordings were stable with good access before the application of ISO or blockers.
Taken together, these results suggest that the observed βAR enhancement of ICa,L occurs mainly via the ACVI isoform. There are 3 main possibilities as to why ACV does not contribute to the observed ICa,L enhancement. First, the ACV isoform may become degraded in the absence of ACVI.7 Second, the ACV isoform may not couple directly to βAR. Third, it is possible that the stimulatory effect of the ACV isoform may be compartmentalized and masked by the action of PDE. Indeed, suppression of β2AR stimulation by PDE was previously documented.15 To directly test the last possibility, we investigated the role of ACV and ACVI separately in the β1AR and β2AR signaling pathways.
β2AR-Mediated Enhancement of ICa,L Signals Mainly via the ACV Isoform
Application of a selective β1AR blocker, CGP-20712A, completely abolished the effect of ISO in WT cardiomyocytes (Figure 2A, top). However, the β2AR-dependent enhancement of ICa,L can be revealed by the application of selective PDE3 and PDE4 blockers, rolipram and cilostamide (Figure 2B and 2D). Similar to WT ventricular myocytes, application of CGP-20712A also abolished the effect of ISO in the ACV KO (Figure 2A, center). In contrast, additional application of PDE blockers failed to reveal the β2AR-dependent increase of ICa,L (Figure 2B and 2D). Administration of CGP-20712A in ACVI KO cardiomyocytes did not change the outcome of ISO stimulation, because there was no increase in ICa,L (Figure 2A, bottom). More important, the addition of PDE blockers in ACVI KO mice revealed the β2AR-dependent enhancement of ICa,L (Figure 2B and 2D), which was not statistically different from the WT group. Taken together, these results suggest that β2AR regulation of ICa,L may signal mainly via the ACV isoform and is under the strong influence of PDE (Figure 2C).
For experiments using PDE blockers, cells were first treated with the blockers for 30 minutes before the addition of ISO. Online Figure IIB shows the time course of the current at baseline, after PDE blockers, and then after ISO in the presence of the blockers.
The significant decrease in the basal ICa,L in ACVI KO and double-KO myocytes was not associated with significant changes in the expression at the protein or transcript level (Online Figure III). Moreover, application of PDE3 and PDE4 blockers alone did not alter ICa,L (Online Figure IIB), suggesting minimal basal phosphorylation of ICa,L. Future experiments are required to further explore the mechanisms underlying the decrease in ICa,L in ACVI KO mice. Nonetheless, the data so far support distinct functional roles of the 2 isoforms of AC in ventricular myocytes.
Two Distinct Populations of β1ARs in Ventricular Myocytes
To directly examine the downstream signaling pathway of β1ARs, we tested the effect of ISO in the presence of 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy] butan-2-ol (ICI)-118 551 (a specific β2AR blocker). In this condition, ISO increased ICa,L in the WT myocytes by 145.8±18.8% at 0 mV (Figure 3A, top). Application of PDE blockers in this condition further potentiated the effect of ISO by 392±63% (Figure 3B and 3D). A similar enhancement of ICa,L in ACV KO myocytes was observed by the application of ISO in the presence of ICI-118 551 (108±16.3% increase in the current). However, in contrast to the WT myocytes, PDE blockers failed to further potentiate the effects of ISO in ACV KO myocytes (Figure 3B and 3D).
As predicted from the results in Figure 1A (bottom), there was no enhancement of ICa,L by ISO in the presence of ICI-118 551 in ACVI KO myocytes. However, the application of PDE blockers revealed a marked increase of ICa,L by 814.2±46% (Figure 3B and 3D). These results suggest that there may exist 2 populations of β1ARs: 1 that is associated with ACVI and another that is associated with ACV and is masked by PDE under basal conditions (Figure 3C). However, PDE inhibition is known to have effects that are independent of βAR stimulation, which may confound the interpretations. Therefore, additional experiments were performed to further test our hypothesis (Figures 4–6).
ACV and ACVI Are the 2 Key Isoforms of AC in Ventricular Myocytes
To test the key isoforms of AC in ventricular myocytes, the effects of ISO on ICa,L were tested in ACV/ACVI KO myocytes in the absence and presence of PDE blockers. There were no observable effects of ISO in these conditions (Online Figure IA–IC), suggesting that ACV and ACVI represent the 2 main isoforms of AC in ventricular myocytes.
Parallel assessment of cAMP levels in ventricular myocytes isolated from ACV/ACV KO and WT mice was performed as previously described.29 The same protocol for patch-clamp recordings was used. Specifically, cells were first incubated with PDE inhibitors for 30 minutes before the addition of β-adrenergic agonist plus PDE inhibitors (Online Figure ID). Results demonstrate a lack of cAMP stimulation in ACV/ACVI KO, consistent with our patch-clamp data.
ACV Isoform Is Compartmentalized Within the t-Tubules Under the Strong Influence of PDE, Whereas ACVI Is Localized Outside the t-Tubules
Our findings thus far support the signaling of β2AR mainly via the ACV isoform. Because previous studies have provided evidence for the localization of β2AR within the t-tubules,19 we tested the hypothesis that the ACV is similarly compartmentalized within the t-tubules. After the detubulation procedure, there were no changes in the effects of ISO on ICa,L in the WT, ACV KO, and ACVI KO myocytes (Online Figure IVA and IVB compared with Figure 1A). However, there was a dramatic decrease in the cell capacitance and the amplitude of ICa,L (Online Figure IVC and IVE). To verify successful detubulation, DI-8-ANEPPS was used to demonstrate the loss of the striation pattern (Online Figure IVD).
More important, after detubulation, the application of PDE blockers failed to reveal the β2AR enhancement of ICa,L current in WT and ACVI KO cardiomyocytes (Figure 4A, right column compared with Figure 2B). In addition, detubulation led to the absence of β1AR enhancement of ICa,L in ACVI KO cardiomyocytes in the presence of PDE blockers (Figure 4B, bottom compared with Figure 3B). In contrast, in WT and ACV KO cardiomyocytes, PDE blockers could potentiate the effect of β1AR stimulation after detubulation (Figure 4B, top and center). Taken together, the data support the notion that β2ARs signal mainly via the ACV isoform. The ACV isoform is compartmentalized within the t-tubules and is under the strong influence of PDE. Moreover, the ACVI isoform appears to be localized outside of the t-tubules and is under weaker influence of PDE.
Finally, from the detubulation experiments, the fraction of L-type Ca2+ channels that are localized in the t-tubule versus the sarcolemmal membrane is estimated to be ≈86% (Online Figure IVE). In addition, we estimated the ratio of Ca2+ channels in the t-tubules that are regulated by β1 versus β2ARs to be 2.4:1 (Online Figure IVE).
Caveolin-3 May Anchor and Localize ACV Within t-Tubules
To investigate the mechanisms for the proposed compartmentalization of ACV within the t-tubules, we tested the hypothesis that caveolin-3, a scaffolding protein, may interact with both ACV and PDE, thus localizing ACV within t-tubules under the strong influence of PDE. Indeed, comparison of the amino acid sequence alignment between the N termini of ACV and ACVI isoforms reveals a putative caveolin-binding domain in the ACV but not ACVI isoform (Figure 5A). The proposed caveolin-binding domain is found to be conserved in both the mouse and human ACV isoform and contains 4 aromatic amino acids, including phenylalanine, at positions 94, 96, and 98 and tryptophan at position 104 in mouse ACV (Figure 5A; Online Figure VA).
To test the hypothesis that the putative caveolin-binding domain may interact with the previously documented caveolin-scaffolding domain in the N terminus of caveolin-3 (Figure 5B), we first modeled the caveolin-3 protein using the Rosetta algorithm with a modification for the transmembrane region (Online Figure VB). Using the docking algorithm, we observed the predicted interactions between the putative caveolin-binding domain and caveolin-scaffolding domain (Online Figure VD and VF). Changing each of the amino acids phenylalanine 94, 96, and 98 and tryptophan 104 in the putative caveolin-binding domain to alanine (Online Figure VE) abolished the interactions in the docking procedure.
Inhibitory Peptides Derived From the Putative Caveolin-Binding Domain or Caveolin-Scaffolding Domain Unmask the Stimulatory Effects of ISO on ICa,L in ACVI KO Mice
We next developed 2 inhibitory peptides (IPs) containing 20 amino acids derived from the N termini of ACV and caveolin-3, which encompass the putative caveolin-binding domain (IP1) and caveolin-scaffolding domain (IP2), respectively. Two control peptides (CPs) were developed by mutating the critical aromatic amino acids (shown in bold in the outlined boxes in Figure 5A and 5B) to alanine.
If caveolin-3 is indeed interacting with ACV and PDE to localize ACV under the strong influence of PDE, we predict that intracellular application of IPs may disrupt the interaction between caveolin-3 and ACV and unmask the stimulatory effects of ISO on ICa,L in ACVI KO myocytes. In fact, this is what we have observed (Figure 5C compared with Figure 1A, bottom). In contrast, application of the 2 CPs (CP1 or CP2) did not affect the response of ICa,L to ISO in ACVI KO myocytes (Figure 5C). Nonetheless, there are other possible interpretations of the results. It is possible that the IPs may result in the relief of the tonic caveolin-3 inhibition on ACV.
Similarly, we also predict a putative caveolin-binding domain in PDE4 isoforms, which seems to be critical for the compartmentalization of ACV isoform (Figure 6A). This sequence is found to be conserved in mouse and human PDE4 across all splice variants (>20). An inhibitory peptide was developed using 20–amino acid sequences encompassing the putative caveolin-binding domain of PDE 4b and 4d (IP3; Figure 6A). A CP was developed by mutating the aromatic amino acid residues to alanine (CP3). Intracellular application of IP3 resulted in the enhancement of ISO on ICa.L in ACVI KO cardiomyocytes (Figure 6B compared with Figure 1A, bottom). In contrast, intracellular application of CP3 did not alter the response of ACVI KO cardiomyocytes to ISO (Figure 6B).
Interactions Between Caveolin-3 and ACV May Help to Compartmentalize the β2AR Signaling
To further test the hypothesis that the interactions between caveolin-3 and ACV may help to compartmentalize the β2AR signaling, we tested the effects of the 2 IPs (IP1 or IP2) on the β2AR regulation of ICa,L in WT ventricular myocytes (Online Figure VIA). Currents were recorded in control and after ISO stimulation in the presence of a selective β1AR blocker (CGP-20712A). Control experiments are shown on the right using CPs (CP1 or CP2). In contrast to Figure 2A, there was a significant enhancement of ICa,L in the presence of the IPs (≈22%–30%; Online Figure VIB and VIC). The enhancement of ICa,L was not observed in ACV KO ventricular myocytes, consistent with data shown using PDE inhibitors (Figure 2B, center).
We further test the effects of PDE4 inhibitor alone on the β2AR stimulation of WT ventricular myocytes (Online Figure VID). PDE4 inhibitor alone was sufficient in relieving the endogenous PDE inhibition on the ICa,L enhancement, consistent with our data using IP3 (Figure 6).
Immunofluorescence Confocal Microscopic Imaging of Caveolin-3 After Detubulation
To properly interpret the results of the detubulation shown in Figure 4, we further confirm that there were no significant changes in the localization of caveolin-3 after the detubulation process (see Online Figure VII; Online Movies). The data further support previous findings that caveolin-3 is localized predominantly in the t-tubules but to a lesser extent on the surface membrane on the surface membrane.12,13
βARs represent one of the most important signaling pathways in the control of heart rates and cardiac contractility. Interest in the βAR pathways stems from their seminal roles in both physiological and pathological conditions.30–32 Indeed, the use of βAR blockers has been shown to be one of the most beneficial therapies for the treatment of heart failure, hypertension, and cardiac arrhythmias.33
Stimulation of L-Type Ca2+ Channels by β1AR and β2AR Is Mediated by Different Isoforms of AC
Functional differences between ACV and ACVI isoforms have not been extensively investigated until recently. The lack of isoform-specific antibodies and the relatively small amount of AC available on the membrane presented technical challenges that have recently been overcome with the development of gene-targeted mouse models. Previous studies have provided evidence that ACV disruption increases longevity and protects against stress.34 In ACVI KO animals,7 there is a significant decrease in βAR reactivity (≈78% reduction). Double KO animals were previously reported to be nonviable; however, we found that double-KO animals could survive despite high mortality rates within the first 3 weeks after birth. After this period, the mice survive without gross abnormalities. This is similar to the limited survival rate of β1AR KO animals.35
Our data support the divergent roles of the 2 isoforms of AC in the heart. In contrast to previous studies, our data suggest the critical roles of both isoforms. There is a complete lack of enhancement of ICa,L by βAR stimulation using ISO in ACVI KO and ACV/ACVI double-KO myocytes. This is in contrast to the WT and ACV KO myocytes. To explain this finding, we considered a possibility that the effects of βAR stimulation via ACV isoform may be masked by PDE. Without PDE blockers, the stimulation of ICa,L from ISO was mediated mostly through β1AR activation (Figures 2A and 3A). Detailed investigation involving the separation of β1AR and β2AR suggests that the effects of β2AR stimulation via the ACV isoform are masked by PDE (as shown in Figure 2). Moreover, the enhancement of ICa,L via the ACV isoform seems to be compartmentalized with both β1AR and β2AR, whereas the enhancement of ICa,L via ACVI is mediated mainly through β1AR. With the detubulation technique, our data suggest that the enhancement effect of ICa,L via ACV is compartmentalized within the t-tubule, whereas the effects of ACVI are coupled to L-type Ca2+ channels, which are localized outside the t-tubules. Indeed, the data are in good agreement with previous findings suggesting that both β1AR and β2AR are located within the t-tubules, whereas only β1AR is present outside the t-tubules.19 Nonetheless, it remains a possibility that the ACV isoform is present in other cellular domains where it might not be coupled to the L-type Ca2+ channels directly. Likewise, the ACVI isoform may be localized but does not couple to the L-type Ca2+ channel in the t-tubules. Future experiments are required to develop antibodies that are isoform specific to directly establish the subcellular localization of the 2 isoforms.
Mechanisms Underlying the Distinct Subcellular Organization and Compartmentalization of AC Isoforms
The different isoforms of AC have similar primary structures containing an N terminus, a 6-transmembrane domain, a C1 portion of the catalytic domain, a second 6-transmembrane domain, and a C2 portion of the catalytic domain. The most striking difference between ACV and ACVI is in the N termini. The N terminus of ACV is much longer than the ACVI terminus, leading us to hypothesize that the specific localization of ACV within the t-tubule may be associated with the N terminus. Because t-tubules are known to contain different lipid contents compared with the outer membrane, we investigated potential differences in the hydrophobicity of the N termini of the 2 isoforms. However, a hydrophobicity plot did not reveal significant differences between the N termini of the ACV and ACVI isoforms.
However, several isoforms of ACs have been shown to be suppressed by the addition of caveolin peptides.36 Because caveolin is a scaffolding protein localized within the t-tubules in addition to the surface sarcolemma, we first tested the hypothesis that the ACV isoform may possess a caveolin-binding domain. Indeed, caveolin-binding domain sequences vary greatly among different proteins, but all share a similar pattern of aromatic alternating with nonaromatic amino acid residues.37 We observe a putative caveolin-binding sequence within the N terminus of ACV. This stretch of amino acids is absent in the ACVI isoform (Figure 5A). IPs generated from the putative binding sequences are able to disrupt the interaction between caveolin and ACV and to unmask the effects of ACV in ACVI KO animals without PDE blockers. Taken together, the data support our notion that this region within the N terminus of ACV is responsible for the association between ACV and caveolin and thus the specific compartmentalization of ACV within the t-tubules.
The location of PDE4 isoforms along the z-line region was previously reported.16 Two isoforms of PDE, PDE4b and PDE4d, have been shown to regulate ICa,L in response to βAR stimulation.16,38,39 We observe a putative caveolin-binding domain within the C termini of the PDE4b and PDE4d isoforms that is highly conserved across species. In fact, the sequence is conserved in >20 different splice variants of PDE4b and PDE4d, suggesting its biological significance. Finally, IPs derived from this putative binding domain unmask the effects of ACV isoform, suggesting that the binding domain is responsible for the interaction between caveolin and PDEs, leading to the compartmentalization of PDE and the ACV isoform within the t-tubules. The schematic representation is presented in Figure 6C.
Potential Clinical Importance and Future Directions
Our findings support the divergent subcellular localization of the 2 main isoforms of AC in ventricular myocytes. The data further suggest that the 2 isoforms represent distinct downstream effector molecules for β1AR versus β2AR signaling pathways. Because of its specialized location, the effects of the ACV isoform are masked by the action of PDE. Finally, the specific protein-protein interactions with the scaffolding protein, caveolin-3, are responsible for the compartmentalization of the ACV isoform, PDE4b, and PDE4d within the t-tubules of cardiomyocytes.
Our data further provide predictions for the disparate function of the 2 isoforms of AC in the heart. Disruption of ACV, PDE4b, and PDE4d could promote βAR signaling. Indeed, an increase in β2AR reactivity has been reported in heart failure, which was explained by the loss of t-tubules in ventricular cardiomyocytes and the redistribution of β2ARs.19 On the basis of our findings, the loss of t-tubules may also be predicted to disrupt the compartmentalization of the ACV-PDE interaction, which may further result in the contribution of β2AR to cardiac toxicity. Finally, our findings help explain the domain organization of βARs in the ventricular cardiomyocytes and suggest that the ACV isoform and the PDE4b and PDE4d isoforms work in concert. Because caveolin-3 represents common scaffolding protein essential for the formation of caveolae in different cell types, our findings may be important not only for cardiac physiology and pathophysiology but also for the organization of cellular compartmentalization in other cell types.
We acknowledge the University of California Davis Health System Confocal Microscopy Facility.
Sources of Funding
This work was supported by a Department of Veteran Affairs Merit Review Grant (I01BX000576) and the National Institutes of Health (NIH) Grants (HL085844, HL085727) to Nipavan Chiamvimonvat and NIH grants (P01 HL066941, HL088426, and HL081741) and a VA Merit Review Award to H. Kirk Hammond. Richard E. Myers and Padmini Sirish are trainees in the NIH T32 HL86350 Training Grant in Basic and Translational Cardiovascular Science to the University of California Davis. This work was also supported by an American Heart Association Grant-in-Aid to Tong Tang.
In March 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.5 days.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.112.300370/-/DC1.
- Nonstandard Abbreviations and Acronyms
- adenylyl cyclase
- ACV KO
- ACV knockout
- ACVI KO
- ACVI knockout
- adrenergic receptor
- control peptide
- L-type Ca2+ current
- inhibitory peptide
- Received October 24, 2012.
- Accepted April 22, 2013.
- © 2013 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Adenylyl cyclase (AC) represents one of the principal effector molecules in the β-adrenergic receptor (βAR) signaling pathway.
AC types 5 (ACV) and 6 (ACVI) are the 2 main isoforms in the heart.
Caveolin-3 is a scaffolding protein, integrating many intracellular signaling molecules in specialized areas called caveolae.
What New Information Does This Article Contribute?
The 2 AC isoforms involved in the regulation of L-type Ca2+ current (ICa,L) in ventricular myocytes show distinct com-partmentalization.
ACV compartmentalized within the t-tubule enhances ICa,L via regulatory signaling restricted by phosphodiesterase.
ACVI enhances ICa,L localized to the outside of t-tubules.
The ACVI isoform is responsible for β1AR signaling–mediated enhancement of ICa,L.
The interaction between caveolin-3 and ACV and phosphodiesterase is responsible for the compartmentalization of ACV signaling.
AC represents one of the principal effector molecules in the βAR signaling pathway. Although ACV and ACVI have been identified as the 2 main isoforms in the heart, it is not known whether these 2 isoforms have distinct subcellular localization. Here, we report that enhancement of ICa,L via ACV isoform is compartmentalized within the t-tubule and is mediated by both β1AR and β2AR. In contrast, the effects of ACVI are coupled to ICa,L channels that are localized outside the t-tubules and are mediated mainly through β1AR. We also found that a specific protein-protein interaction with caveolin-3 is responsible for the compartmentalization of the ACV isoform and phosphodiesterase 4b and 4d within the t-tubules of cardiomyocytes. Hence, loss of t-tubules, which has been reported in heart failure, could disrupt the compartmentalization of ACV and phosphodiesterase, resulting in an increase in the contribution of βAR to cardiac toxicity.