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Role of α2-Adrenoceptor Subtypes in Suppression of L-Type Ca2+ Current in Mouse Cardiac Myocytes.

ABSTRACT: Sarcolemmal α2 adrenoceptors (α2-AR), represented by α2A, α2B and α2C isoforms, can safeguard cardiac muscle under sympathoadrenergic surge by governing Ca2+ handling and contractility of cardiomyocytes. Cardiomyocyte-specific targeting of α2-AR would provide cardiac muscle-delimited stress control and enhance the efficacy of cardiac malfunction treatments. However, little is known about the specific contribution of the α2-AR subtypes in modulating cardiomyocyte functions. Herein, we analyzed the expression profile of α2A, α2B and α2C subtypes in mouse ventricle and conducted electrophysiological antagonist assay evaluating the contribution of these isoforms to the suppression of L-type Ca2+ current (ICaL). Patch-clamp electro-pharmacological studies revealed that the α2-agonist-induced suppression of ICaL involves mainly the α2C, to a lesser extent the α2B, and not the α2A isoforms. RT-qPCR evaluation revealed the presence of adra2b and adra2c (α2B and α2C isoform genes, respectively), but was unable to identify the expression of adra2a (α2A isoform gene) in the mouse left ventricle. Immunoblotting confirmed the presence only of the α2B and the α2C proteins in this tissue. The identified α2-AR isoform-linked regulation of ICaL in the mouse ventricle provides an important molecular substrate for the cardioprotective targeting.

SUBMITTER: Evdokimovskii EV

PROVIDER: S-EPMC8072751 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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Role of α2-Adrenoceptor Subtypes in Suppression of L-Type Ca<sup>2+</sup> Current in Mouse Cardiac Myocytes.

Evdokimovskii Edward V EV Jeon Ryounghoon R Park Sungjo S Pimenov Oleg Y OY Alekseev Alexey E AE

International journal of molecular sciences 20210416 8

Sarcolemmal α2 adrenoceptors (α2-AR), represented by α2A, α2B and α2C isoforms, can safeguard cardiac muscle under sympathoadrenergic surge by governing Ca<sup>2+</sup> handling and contractility of cardiomyocytes. Cardiomyocyte-specific targeting of α2-AR would provide cardiac muscle-delimited stress control and enhance the efficacy of cardiac malfunction treatments. However, little is known about the specific contribution of the α2-AR subtypes in modulating cardiomyocyte functions. Herein, we ...[more]

PMID: 33923625

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Project description:The effects of several phosphodiesterase (PDE) inhibitors on the L-type Ca current (I(Ca)) and intracellular cyclic AMP concentration ([cAMP]i) were examined in isolated rat ventricular myocytes. The presence of mRNA transcripts encoding for the different cardiac PDE subtypes was confirmed by RT-PCR. IBMX (100 microM), a broad-spectrum PDE inhibitor, increased basal I(Ca) by 120% and [cAMP]i by 70%, similarly to a saturating concentration of the beta-adrenoceptor agonist isoprenaline (1 microM). However, MIMX (1 microM), a PDE1 inhibitor, EHNA (10 microM), a PDE2 inhibitor, cilostamide (0.1 microM), a PDE3 inhibitor, or Ro20-1724 (0.1 microM), a PDE4 inhibitor, had no effect on basal I(Ca) and little stimulatory effects on [cAMP]i (20-30%). Each selective PDE inhibitor was then tested in the presence of another inhibitor to examine whether a concomitant inhibition of two PDE subtypes had any effect on I(Ca) or [cAMP]i. While all combinations tested significantly increased [cAMP]i (40-50%), only cilostamide (0.1 microM)+ Ro20-1724 (0.1 microM) produced a significant stimulation of I(Ca) (50%). Addition of EHNA (10 microM) to this mix increased I(Ca) to 110% and [cAMP]i to 70% above basal, i.e. to similar levels as obtained with IBMX (100 microM) or isoprenaline (1 microM). When tested on top of a sub-maximal concentration of isoprenaline (1 nM), which increased I(Ca) by (approximately 40% and had negligible effect on [cAMP]i, each selective PDE inhibitor induced a clear stimulation of [cAMP]i and an additional increase in I(Ca). Maximal effects on I(Ca) were approximately 8% for MIMX (3 microM), approximately 20% for EHNA (1-3 microM), approximately 30% for cilostamide (0.3-1 microM) and approximately 50% for Ro20-1724 (0.1 microM). Our results demonstrate that PDE1-4 subtypes regulate I(Ca) in rat ventricular myocytes. While PDE3 and PDE4 are the dominant PDE subtypes involved in the regulation of basal I(Ca), all four PDE subtypes determine the response of I(Ca) to a stimulus activating cyclic AMP production, with the rank order of potency PDE4>PDE3>PDE2>PDE1.

Project description:Junctophilins (JPs), a protein family of the junctional membrane complex, maintain the close conjunction between cell surface and intracellular membranes in striate muscle cells mediating the crosstalk between extracellular Ca2+ entry and intracellular Ca2+ release. The small-conductance Ca2+ -activated K+ channels are activated by the intracellular calcium and play an essential role in the cardiac action potential profile. Molecular mechanisms of regulation of the SK channels are still uncertain. Here, we sought to determine whether there is a functional interaction of junctophilin type 2 (JP2) with the SK channels and whether JP2 gene silencing might modulate the function of SK channels in cardiac myocytes. Association of JP2 with SK2 channel in mouse heart tissue as well as HEK293 cells was studied using in vivo and in vitro approaches. siRNA knockdown of JP2 gene was assessed by real-time PCR. The expression of proteins was analysed by Western blotting. Ca2+ -activated K+ current (IK,Ca ) in infected adult mouse cardiac myocytes was recorded using whole-cell voltage-clamp technique. The intracellular Ca2+ transient was measured using an IonOptix photometry system. We showed for the first time that JP2 associates with the SK2 channel in native cardiac tissue. JP2, via the membrane occupation and recognition nexus (MORN motifs) in its N-terminus, directly interacted with SK2 channels. A colocalization of the SK2 channel with its interaction protein of JP2 was found in the cardiac myocytes. Moreover, we demonstrated that JP2 is necessary for the proper cell surface expression of the SK2 channel in HEK293. Functional experiments indicated that knockdown of JP2 caused a significant decrease in the density of IK,Ca and reduced the amplitude of the Ca2+ transient in infected cardiomyocytes. The present data provide evidence that the functional interaction between JP2 and SK2 channels is present in the native mouse heart tissue. Junctophilin 2, as junctional membrane complex (JMC) protein, is an important regulator of the cardiac SK channels.

Dataset Information

Role of α2-Adrenoceptor Subtypes in Suppression of L-Type Ca2+ Current in Mouse Cardiac Myocytes.

Publications

Role of α2-Adrenoceptor Subtypes in Suppression of L-Type Ca<sup>2+</sup> Current in Mouse Cardiac Myocytes.

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