|Home | About | Journals | Submit | Contact Us | Français|
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.
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 cells (Cn-NFAT) signaling cascade that promotes pathological hypertrophy.
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, almost all of the Ca2+ influx induced NFAT nuclear translocation, but did not reduce myocyte contractility.
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 failure1,2, but the source of this “hypertrophic” Ca2+ is still 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) pathway2, 3, the calmodulin-dependent protein kinase (CaMKII) pathway , and the protein kinase C (PKC) pathway5. The source of the Ca2+ that activates each of4 these signaling cascades is still not well known. Potential sources include L-6, 7 and T-8, 9 type Ca2+ channels and transient receptor potential (TRP) channels10.
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 (ECC) and regulation of gene expression1. Myocyte LTCCs are concentrated in T-tubular membranes where they are organized in junctional complexes with ryanodine receptors (RyR2) on the SR1 to form a signaling microdomain involved in Ca2+-induced-SR Ca2+-release (CICR). This microdomain is the source of “contractile” [Ca2+].
Not all LTCCs are involved in CICR. A small fraction appears to be harbored in caveolae11, 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 microdomains11. 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 LTCCs12. Expressing wild type Rem in cardiac myocytes blocked ICa-L and this inhibition required a c-terminal membrane-docking domain13. 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, did not alter myocyte contractility, but 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 WT-Rem, Rem1-265, Rem1-265-Cav, and/or NFATc3-GFP17, all at a multiplicity of infection (MOI) 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 techniques16.
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 (H). 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/caveolae targeting of LTCCs, eNOS 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 purified16. 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 IP was then re-immunoprecipitated for Cav1.2 (Figure 1C). Densitometric analysis (n=3) showed that 26.2+/-12.7% of LTCCs resides 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-immunoprecipitated 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.
WT-Rem almost fully eliminated the L-type Ca2+ channel current (ICa,L) in AFLVMs at an MOI of 100 (Figure 2A). Rem1-265 had no significant effect on ICa,L. Rem1-265-Cav caused a small inhibition (about 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 catecholamines12. AFLVMs infected with Rem1-265-Cav responded normally to Isoproterenol (Figure 2A) while those infected with WT-Rem failed to respond (not shown). Myocytes infected with WT-Rem had markedly reduced fractional shortening (1.1+/-0.1% resting cell length) while 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 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 (ECC).
Myocyte Cav-3 membrane domains are thought to contain both LTCCs and β2-adrenergic receptors11. The β2-adrenergic receptor specific agonist Zinterol in the presence of the β1-adrenergic receptor specific antagonist CGP 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.
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 SR Ca2+ release9 and NFATc3-GFP is localized to the cytoplasm17. NFAT localization (cytoplasmic versus nuclear) was measured before (Figure 4A and C) and after (Figure 4B and 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 above) but more than 90% of NFAT nuclear translocation was inhibited (Figure 4E and F). These results were confirmed in experiments in which bath Ca2+ was raised to 4 mM, 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 (CaM) 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 process6.
The sources of “hypertrophic” [Ca2+] to activate Cn-NFAT signaling are not clearly defined. There is some evidence for a role for the LTCC6, 7, however, there is also evidence for Ca2+ entry through T-type Ca2+ channels (TTCC)8, 9 and TRP channels10. The role of TTCCs is controversial since some studies suggest that this pathway is anti-hypertrophic8 rather than pro-hypertrophic9. The observation that excess Ca2+ influx through TTCCs channels is anti-hypertrophic8 suggests that Ca2+ influx per se is not pro- or anti-hypertrophic. 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 Cn18. These Cav-3 localized LTCCs do not participate in ECC 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 others have found within T-tubules do not participate in ECC19.
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 function1. Our results suggest that nature uses a subpopulation of these channels, housed away from ECC 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.
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.
Sources of funding: SRH-NIH Grants R01HL089312, T32HL091804, P01HL091799, R37HL033921, JDM-NIH Grants R01HL089312, R01HL62927, P01HL080101 CAM-AHA Pre-Doctoral Fellowship, RNC-NIH Post-Doctoral Fellowship
Disclosures: The Authors have no disclosures
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.