A Caveolae-Targeted L-Type Ca2+ Channel Antagonist Inhibits Hypertrophic Signaling Without Reducing Cardiac ContractilityNovelty and Significance
Rationale: The source of Ca2+ to activate pathological cardiac hypertrophy is not clearly defined. Ca2+ influx through the L-type Ca2+ channels (LTCCs) determines “contractile” Ca2+, which is not thought to be the source of “hypertrophic” Ca2+. However, some LTCCs are housed in caveolin-3 (Cav-3)–enriched signaling microdomains and are not directly involved in contraction. The function of these LTCCs is unknown.
Objective: To test the idea that LTCCs in Cav-3–containing signaling domains are a source of Ca2+ to activate the calcineurin–nuclear factor of activated T-cell signaling cascade that promotes pathological hypertrophy.
Methods and Results: We developed reagents that targeted Ca2+ channel-blocking Rem proteins to Cav-3–containing membranes, which house a small fraction of cardiac LTCCs. Blocking LTCCs within this Cav-3 membrane domain eliminated a small fraction of the LTCC current and almost all of the Ca2+ influx-induced NFAT nuclear translocation, but it did not reduce myocyte contractility.
Conclusions: We provide proof of concept that Ca2+ influx through LTCCs within caveolae signaling domains can activate “hypertrophic” signaling, and this Ca2+ influx can be selectively blocked without reducing cardiac contractility.
Cardiovascular diseases increase cardiac systolic stress and cause myocyte hypertrophy. Increases in Ca2+ influx during pathological stress maintain pump function and activate signaling pathways that produce pathological hypertrophy and heart failure,1,2 but the source of this “hypertrophic” Ca2+ still is not clearly defined.
Increases in Ca2+ influx are essential for the activation of pathological hypertrophic signaling, including the calcineurin–nuclear factor of activated T-cells (Cn-NFAT) pathway,2,3 the calmodulin-dependent protein kinase pathway,4 and the protein kinase C pathway.5 The source of the Ca2+ that activates each of these signaling cascades is still not well-known. Potential sources include L-type6,7 and T-type8,9 Ca2+ channels and transient receptor potential channels.10
In cardiac myocytes, voltage-dependent L-type Ca2+ channels (LTCC, Cav1.2) are the predominant Ca2+ entry pathway and are essential for excitation–contraction coupling and regulation of gene expression.1 Myocyte LTCCs are concentrated in T-tubular membranes, where they are organized in junctional complexes with ryanodine receptors on the sarcoplasmic reticulum1 to form a signaling microdomain involved in Ca2+-induced sarcoplasmic reticulum Ca2+ release. This microdomain is the source of “contractile” [Ca2+].
Not all LTCCs are involved in Ca2+-induced sarcoplasmic reticulum Ca2+ release. A small fraction appears to be harbored in caveolae,11 which are highly specialized membrane regions that are stabilized by the scaffolding protein caveolin. Caveolin-3 is the major caveolin expressed in the heart and it functions to help organize local signaling microdomains.11 The function of caveolae-based LTCCs are unknown and are the topic of this study.
Our studies test the hypothesis that “hypertrophic” Ca2+ enters the myocyte through LTCCs localized in Cav-3–based signaling microdomains and that this “signaling” Ca2+ is distinct from “contractile” Ca2+. This idea was examined with a novel LTCC blocker that selectively traffics to caveolae, where it inhibits LTCCs within this signaling microdomain. Our results show that this Cav-3–localized LTCC antagonist can block “hypertrophic” Ca2+ without altering contractility. The caveolae targeted LTCC blocker was generated by molecular modification of Rem, a member of the RGK GTPase family that is known to potently inhibit LTCCs.12 Expressing wild-type Rem in cardiac myocytes blocked L-type Ca2+ channel current (ICa-L), and this inhibition required a c-terminal membrane-docking domain.13 Truncation of this membrane-docking domain (Rem1-265) resulted in the inability of Rem to traffic to the membrane and to inhibit ICa-L. To specifically block caveolae-based LTCCs, the membrane-binding motif of native Rem was deleted and replaced with a canonical caveolin-targeting domain sequence14 to create Rem1-265-Cav. Rem1-265-Cav targeted selectively to Cav-3 membranes and did not alter myocyte contractility, but it blocked Ca2+-influx–mediated activation of Cn-NFAT signaling. These data strongly support the hypothesis that LTCCs housed in Cav-3–containing microdomains is a source of “hypertrophic” Ca2+.
Adult feline left ventricular myocytes (AFLVMs) were isolated15 and adenoviral gene transfer was used to introduce wild-type (WT)-Rem, Rem1-265, Rem1-265-Cav, or NFATc3-GFP,17 all at a multiplicity of infection of 100. Myocytes were cultured for a period of up to 4 days. Localization of AdRem constructs was confirmed using standard sucrose gradient fractionation and caveolin-3 immunoisolation techniques.16
Rem1-265-Cav Localizes to Caveolae
Native Rem was not detected in plasma membrane (PM) from AFLVMs. After adenoviral infection, WT-Rem was found in all AFLVM membrane fractions, consistent with the idea that it is in all PM domains. The Rem1-265 peptide lacking the membrane association motif remained in the general cell homogenate. Rem1-265-Cav localized to PM specifically within caveolin-containing lipid rafts (Figure 1A), verifying target specificity of the peptide containing the Cav-binding motif. Rem1-265-Cav did not alter the normal raft or caveolae targeting of LTCCs, endothelial nitric oxide synthase, and caveolin-3, which demonstrates that Rem1-265-Cav does not displace molecules normally found in caveolae (Figure 1A). To prove that Rem1-265-Cav specifically localizes to caveolae, rather than lipid rafts in general, caveolae organelles were immunoaffinity-purified.16 Similar to our observation in raft membranes, only Rem1-265-Cav was seen in association with caveolin-3 (Figure 1B). These data show that Rem1-265-Cav specifically traffics to Cav-3 membrane microdomains.
To further assess what fraction of LTCCs localizes to caveolae membrane domains, PM samples were purified from AFLVMs and subjected to immunoaffinity isolation with anti-Cav-3 (Figure 1C). The unbound fraction from the Cav-3 immunoprecipitation (IP) was then re-IM for Cav1.2 (Figure 1C). Densitometric analysis (n=3) showed that 26.2%±12.7% of LTCCs reside within caveolae. To estimate the fraction of caveolae that contain LTCCs, AFLVM PM preparations were immunoprecipitated with an antibody for Cav1.2 (LTCC). The resulting unbound fraction was then re-IM for Cav-3 and the intensity of the bands in both fractions were compared. Only 13.4%±10.1% (n=3) of caveolae PM contained LTCCs.
Rem1-265-Cav Blocks a Small Fraction of ICa,L and Has No Effect on Contractility
WT-Rem almost fully eliminated the L-type Ca2+ channel current (ICa,L) in AFLVMs at an multiplicity of infection of 100 (Figure 2A). Rem1-265 had no significant effect on ICa,L. Rem1-265-Cav caused a small inhibition (approximately 15%) of ICa,L (Figure 2A). The smaller ICa,L block with Rem1-265-Cav versus WT-Rem suggests that only a small fraction of LTCCs are localized to Cav-3–signaling microdomains.
Inhibition of ICa,L with WT-Rem eliminates LTCC regulation by catecholamines.12 AFLVMs infected with Rem1-265-Cav responded normally to isoproterenol (Figure 2A), whereas those infected with WT-Rem did not respond (not shown). Myocytes infected with WT-Rem had markedly reduced fractional shortening (1.1%±0.1% resting cell length), whereas those infected with truncated Rem1-265 (5.0%±0.5%) and Rem1-265-Cav (4.4%±0.4%) had contractions that were not significantly smaller than those of controls (4.7%±0.5%;Figure 2B). [Ca2+]i transients were also unaffected by Rem1-265-Cav (Figure 2C). Collectively, these data support the idea that Rem1-265-Cav blocks a small fraction of Cav-3–associated LTCCs without significantly effecting myocyte excitation–contraction coupling.
Rem1-265-Cav Inhibits the β2-Adrenergic Regulation of LTCCs
Myocyte Cav-3 membrane domains are thought to contain both LTCCs and β2-adrenergic receptors.11 The β2-adrenergic receptor-specific agonist Zinterol in the presence of the β1-adrenergic receptor-specific antagonist CGP 20712 A increased ICa,L and fractional shortening in AFLVMs. This effect was inhibited by Rem1-265-Cav (Figure 3), suggesting that Rem1-265-Cav can selectively block Ca2+ entry through β2-adrenergic receptor-regulated LTCCs within Cav-3–signaling microdomains.
Rem1-265-Cav Inhibits NFAT Translocation to the Nucleus
AFLVMs infected with AdNFATc3-GFP or AdNFATc3-GFP and AdRem1-265-Cav were electrically quiescent in culture. AFLVMs maintain low levels of cytosolic [Ca2+] with little or no spontaneous sarcoplasmic reticulum Ca2+ release9 and NFATc3-GFP is localized to the cytoplasm.17 NFAT localization (cytoplasmic vs nuclear) was measured before (Figure 4A, C) and after (Figure 4B, D) 1-Hz pacing for 1 hour. Pacing caused NFAT to translocate from the cytoplasm to the nucleus in control AFLVMs (Figure 4B). AFLVMs infected with Rem1-265-Cav had normal contractions and Ca2+ transients (shown), but more than 90% of NFAT nuclear translocation was inhibited (Figure 4E, F). These results were confirmed in experiments in which bath Ca2+ was increased to 4 mmol/L, which also induced NFAT to translocate to the nucleus in control cells. Rem1-265-Cav inhibited NFAT translocation under these conditions (data not shown).
Ca2+-calmodulin–dependent activation of Cn-NFAT2,3 produces pathological hypertrophy. Activation of this signaling cascade requires an increase in myocyte [Ca2+]. “Contractile” [Ca2+] does not appear to activate this hypertrophic process.6
The sources of “hypertrophic” [Ca2+] to activate Cn-NFAT signaling are not clearly defined. There is some evidence for a role for the LTCC;6,7 however, there is also evidence for Ca2+ entry through T-type Ca2+ channels8,9 and transient receptor potential channels.10 The role of T-type Ca2+ channels is controversial because some studies suggest that this pathway is antihypertrophic8 rather than prohypertrophic.9 The observation that excess Ca2+ influx through T-type Ca2+ channels is antihypertrophic8 suggests that Ca2+ influx per se is not prohypertrophic or antihypertrophic. Rather, the pathway for Ca2+ influx and possibly the localization of this pathway appear to be critical.
The present experiments suggest that a small number of LTCCs are localized in a fraction of Cav-3–containing membranes to form a signaling microdomain that can activate Cn-NFAT signaling. Our results are consistent with a recent study suggesting that a subpopulation of LTCCs in Cav-3–containing membranes are housed with AKAP150, which binds Cn.18 These Cav-3–localized LTCCs do not participate in excitation—contractoin coupling or the regulation of “contractile” Ca2+. Our Cav-3–targeting strategy was able to selectively traffic an LTCC-blocking Rem protein (Rem1-265-Cav) to this signaling domain to block hypertrophic signaling without significantly reducing contraction or β1AR regulation of contractility. Our results suggest that any LTCCs associated with the small amount of Cav-3 that others have found within T-tubules do not participate in excitation–contraction coupling.19
Our results also show that not all Cav-3–containing membrane domains contain LTCCs, suggesting that hypertrophic signaling may take place in highly specialized signaling complexes. The signaling partners within these domains and their regulation in health and disease needs to be determined in future studies.
LTCCs are the major Ca2+ influx pathway in the adult mammalian heart, and these channels are regulated biosensors that determine myocyte contractility and cardiac pump function.1 Our results suggest that nature uses a subpopulation of these channels, housed away from excitation–contraction coupling signaling domains, as a source of Ca2+ to modulate myocyte size when the heart is subjected to stress. Selective block of this Ca2+ influx pathway by the novel reagents developed in this study might provide an approach to reduce pathological hypertrophy without reducing cardiac contractility.
Sources of Funding
S.R.H. received NIH grants R01HL089312, T32HL091804, P01HL091799, and R37HL033921. J.D.M. received NIH grants R01HL089312, R01HL62927, and P01HL080101. C.A.M. received an AHA predoctoral fellowship. R.N.C. received an NIH postdoctoral fellowship. V.R. received NIH R01HL086551.
In December 2011, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.29 days.
Brief UltraRapid Communications (BURCs) are designed to be a format for manuscripts that are of outstanding interest to the readership, report definitive observations, but have a relatively narrow scope. Less comprehensive than Regular Articles but still scientifically rigorous, BURCs present seminal findings that have the potential to open up new avenues of research. A decision on BURCs is rendered within 7 days of submission.
This manuscript was sent to Mark Sussman, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
↵* These authors contributed equally to this work.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.111.264028/-/DC1.
Non-standard Abbreviations and Acronyms
- adult feline left ventricular myocyte
- L-type calcium channel
- nuclear factor of activated T cells
- Received December 31, 2011.
- Revision received January 17, 2012.
- Accepted January 25, 2012.
- © 2012 American Heart Association, Inc.
- Chen X,
- Zhang X,
- Kubo H,
- Harris DM,
- Mills GD,
- Moyer J,
- Berretta R,
- Potts ST,
- Marsh JD,
- Houser SR
- Eder P,
- Molkentin JD
- Balijepalli RC,
- Foell JD,
- Hall DD,
- Hell JW,
- Kamp TJ
- Xu X,
- Marx SO,
- Colecraft HM
- Correll RN,
- Pang C,
- Finlin BS,
- Dailey AM,
- Satin J,
- Andres DA
- Couet J,
- Li S,
- Okamoto T,
- Ikezu T,
- Lisanti MP
- Carlile-Klusacek M,
- Rizzo V
- MacDonnell SM,
- Weisser-Thomas J,
- Kubo H,
- Hanscome M,
- Liu Q,
- Jaleel N,
- Berretta R,
- Chen X,
- Brown JH,
- Sabri AK,
- Molkentin JD,
- Houser SR
- Nichols CB,
- Rossow CF,
- Navedo MF,
- Westenbroek RE,
- Catterall WA,
- Santana LF,
- McKnight GS
Novelty and Significance
What is Known?
Pathological cardiac stressors such as hypertension and myocardial infarction activate signaling cascades that lead to cardiac myocyte hypertrophy.
Increases in myocyte [Ca2+]i are essential to initiate hypertrophic signaling through the cytoplasmic protein phosphatase Calcineurin (Cn).
The cytoplasmic [Ca2+]i transient is initiated by Ca2+ influx through L-type Ca2+ channels (LTCCs) and drives cardiac contraction. This “contractile” [Ca2+]i does not appear to be the source of [Ca2+] to initiate pathological hypertrophy.
What New Information Does This Article Contribute?
A small population of LTCCs is housed in Caveolin (Cav)-3–containing membranes (Caveolae) and is not involved in the regulation of contractile [Ca2+]i.
An LTCC-blocking protein can be trafficked to Cav-3–containing membranes to selectively block LTCCs in this signaling microdomain.
Block of Ca2+ entry through LTCCs within caveolae abolished activation of the Cn signaling pathway that is known to cause pathological hypertrophy.
There is strong epidemiological evidence to suggest that pathological cardiac hypertrophy predisposes patients to adverse cardiac events and to heart failure. Increases in [Ca2+]i are necessary to activate pathological hypertrophy, but the source of this Ca2+ appears to be distinct from the [Ca2+]i that is required for cardiac contraction. This study shows that a small population of LTCCs in a specialized signaling microdomain can be a source of Ca2+ to activate hypertrophic signaling. We developed a novel reagent that selectively blocks “hypertrophic” Ca2+ entry through Cav-3–based channels without blocking the LTCCs that induce and regulate “contractile” Ca2+. These experiments suggest that selective block of LTCCs in Cav-3–based signaling microdomains could reduce pathological hypertrophy without causing adverse negative inotropic effects.