Adenylyl cyclase subtype-specific compartmentalization: differential regulation of L-type Ca2+ current in ventricular myocytes - PubMed (original) (raw)

. 2013 Jun 7;112(12):1567-76.

doi: 10.1161/CIRCRESAHA.112.300370. Epub 2013 Apr 22.

Richard E Myers, Hyo Jeong Kim, Ryan L Woltz, Padmini Sirish, James P Heiserman, Ning Li, Anil Singapuri, Tong Tang, Vladimir Yarov-Yarovoy, Ebenezer N Yamoah, H Kirk Hammond, Nipavan Chiamvimonvat

Affiliations

Adenylyl cyclase subtype-specific compartmentalization: differential regulation of L-type Ca2+ current in ventricular myocytes

Valeriy Timofeyev et al. Circ Res. 2013.

Abstract

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 Ca(2+) 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.

Keywords: L-type Ca2+ current; adenylyl cyclase type 5; adenylyl cyclase type 6; adrenergic receptor; calcium channel; ventricular myocytes.

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Figures

Figure 1

Figure 1. β-AR enhances _I_Ca,L in mouse ventricular cardiomyocytes via the activation of ACVI

(a) Representative _I_Ca,L recorded from ventricular myocytes isolated from WT, ACV KO, and ACVI KO, respectively. Current traces were elicited using a voltage step of 0 mV for 500 ms from a holding potential of -55 mV at baseline (blue) and after 20 minutes of ISO (red). The corresponding currentvoltage (IV) relations elicited using a family of voltage steps from -40 to +60 mV from a holding potential of -55 mV are shown to the right. (b) Schematic representation of the known regulation of _I_Ca,L by β-AR stimulation. (c) Summary data of _I_Ca,L density (in pA/pF) from the three groups of mice (n=8 for each group, *P<0.05).

Figure 2

Figure 2. ACV isoform is critical for β2AR enhancement of _I_Ca,L in the mouse ventricular myocytes

(a) Representative _I_Ca,L recorded from ventricular myocytes isolated from WT, ACV KO, and ACVI KO, respectively. Current traces were elicited using a voltage step of 0 mV for 500 ms from a holding potential of -55 mV at baseline (blue) and after 20 minutes of ISO (red). β1ARs were blocked by CGP-20712A (CGP). No significant effect of β2AR stimulation was observed. (b) Application of a PDE3 blocker (rolipram, Rp) and a PDE4 blocker (cilostamide, CI) revealed the effect of β2AR stimulation only in WT and ACVI KO animals. (c) Schematic representation of the experimental results suggesting that ACV is localized within the same compartment as β2ARs and PDE shown in the box as outlined, separated from β1ARs. (d) Summary data of the percentages of _I_Ca,L enhancement by β2AR stimulation in the three groups of mice with and without PDE blockers (n=7-8 for each group, *P<0.05).

Figure 3

Figure 3. β1ARs mediate the enhancement effects on _I_Ca,L via both ACV and ACVI isoforms

(a) Representative _I_Ca,L recorded from ventricular myocytes isolated from WT, ACV KO, and ACVI KO, respectively. Current traces were elicited using a voltage step of 0 mV for 500 ms from a holding potential of -55 mV at baseline (blue) and after 20 minutes of ISO (red). β2-ARs were blocked by ICI- 118,551 (ICI). No β1AR stimulation of the _I_Ca,L was observed in the ACVI KO group. (b) Application of a PDE3 blocker (Rp) and a PDE4 blocker (CI) revealed the stimulatory effect of β1AR in the ACVI KO mice and further enhanced the effects of β1AR in the WT mice. (c) Schematic representation of the experimental results which suggest that β1ARs mediate the enhancement effects on _I_Ca,L via both ACV and ACVI isoforms. Moreover, only the effect through the ACV isoform is restricted by PDE. (d) Summary data of the percentages of _I_Ca,L enhancement by β1AR stimulation in the three groups of mice with and without PDE blockers (n=6-8 for each group, *P<0.05).

Figure 4

Figure 4. Detubulation abolished the enhancement effects of β2AR stimulation on _I_Ca,L via ACV isoform

(a) Representative _I_Ca,L recorded from ventricular myocytes after detubulation from WT, ACV KO, and ACVI KO, respectively. Current traces were elicited using a voltage step of 0 mV for 500 ms from a holding potential of -55 mV at baseline (blue) and after 20 minutes of ISO (red) in the presence of a β1AR blocker (CGP) without PDE blockers (left column) and with PDE blockers (right column). Compared to Figure 2, after detubulation, PDE blockers failed to enhance _I_Ca,L in the WT or ACVI KO mice. (b) Similar experiments conducted in the presence of a β2AR blocker (ICI) without PDE blockers (left column) and with PDE blockers (right column). Compared to Figure 3, after detubulation, PDE blockers failed to enhance _I_Ca,L in the ACVI KO group. (c) Schematic representation of the experimental results which support the compartmentalization of ACV signaling within the t-tubule. (d) Summary data of the percentages of _I_Ca,L enhancement in the three groups of mice with and without PDE blockers after detubulation in the presence of β1AR or β2AR blockers (n=6-8 for each group, *P<0.05).

Figure 5

Figure 5. Caveolin-3 interacts and anchors ACV isoform within t-tubules in ventricular myocytes

(a, b) Amino acid sequence alignment of the N-termini of human (Hs) and mouse (Mm) ACV and ACVI isoforms in (a) and the N-termini of human (Hs) and mouse (Mm) caveolin-3 (CAV3) in (b). Two inhibitory peptides were generated encompassing the conserved region of ACV (outlined in the box, IP1) and the scaffolding domain in CAV3 (outlined in the box, IP2), respectively. The corresponding control peptides (CP1, CP2) were generated by mutating the aromatic amino acids (shown in bold and underlined in a & b) into alanine. Note that the ACVI isoform does not contain the putative caveolin-binding sequence. (c) Representative _I_Ca,L recorded from ACVI KO in the presence of the inhibitory and control peptides in control (blue) and after 20 minutes ISO application (red). Both inhibitory peptides (IP1 and IP2) revealed the significant stimulatory effects of ISO. Summary data of the percentages of _I_Ca,L enhancement in ACVI KO cardiomyocytes in the presence of the inhibitory or control peptides (n=6 for each group, *P<0.05). (d) Schematic representation of the experimental results which suggest a caveolin binding domain on N-terminus of the ACV isoform. Caveolin-3 interacts and anchors the ACV isoform within the t-tubules of the cardiac myocytes. A pink ellipse represents the caveolin scaffolding domain.

Figure 6

Figure 6. Cavelin 3 associates with PDE and localizes PDE to ACV compartment within t-tubules in ventricular myocytes

(a) Amino acid sequence alignment of human (Hs) and mouse (Mm) PDE4b and d. Both isoforms contain putative caveolin-binding domains. A 20-amino-acid inhibitory peptide was generated encompassing the putative caveolin-binding domain (outlined in the box, IP3). A corresponding control peptide (CP3) was generated by mutating the aromatic amino acids (shown in bold and underlined) to alanine. (b) Representative _I_Ca,L recorded from ACVI KO cardiomyocytes in the presence of IP3 (left) and CP3 (right) in control (blue) and after 20 minutes ISO application (red). Lower panel shows the summary data of the percentages of _I_Ca,L enhancement in ACVI KO cardiomyocytes in the presence IP3 compared to CP3 (n=6 for each group, *P<0.05). (c) Schematic representation of the experimental results showing the association between caveolin-3 and PDE and the compartmentalization of PDE and ACV isoform within the t-tubules in ventricular myocytes.

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