Functional LTCC-β ₂ AR Complex Needs Caveolin-3 and Is Disrupted in Heart Failure

Figures

• 

  Figure 1.. L-type Ca2+ channels (LTCCs) response to isoproterenol on control and failing cardiomyocytes. A, 10×10 µm representative scanning ion conductance microscopy topographical images of control cardiomyocytes showing the position of the pipette on the TT or Crest microdomain. B, Representative single LTCC traces at −6.7 mV. C, Summary graph of the Po from control and failing cardiomyocytes in TT and Crest before and after 1 µM ISO application (n=channels/cells/animals TT control 21/8/6, Crest control 17/7/5, TT failing 8/5/4, Crest failing 15/7/7, P values by Wilcoxon matched-pairs signed rank test). D, Representative ICa,L traces of a control cell before and after 1 µM ISO application. E, I/V graph of control cells, inset showing the differences at 0 mV (n=16/5, *P<0.05; P value by Wilcoxon matched-pairs signed rank test). F, Representative ICa,L traces of a failing cell before and after 0.1 µM ISO application. G, I/V graph of failing cells, inset showing the differences at 0 mV (n=15/4, P value by Wilcoxon matched-pairs signed rank test).

• 

  Figure 2.. Protein phosphorylation analysis by WB and ICa,L response to beta-2 adrenergic receptor (β1AR) or beta-2 adrenergic receptor (β2AR) stimulation. A, Representative WBs of control and failing samples. B, Summary graph of densitometry analysis of pPLN for the PKA site (Ser16) vs total PLN, normalized to α-actinin from control (n=8) and failing (n=4) rat cardiomyocytes isolations (P values by Mann-Whitney U test). C, Summary graph of densitometry analysis of pPLN for CaMKII (Ca2+/calmodulin-dependent protein kinase II) site (Thr17) vs total PLN, normalized to GAPDH from control (n=6) and failing (n=4) rat cardiomyocytes isolations (P values by Mann-Whitney U test). D, Representative WBs of control cells under no stimulation, under β1AR stimulation, or under β2AR stimulation. E, Summary graph of densitometry analysis of pPLN (Ser16) vs total PLN, normalized to α-actinin from control cardiomyocytes after β1AR or β2AR stimulation (n=6, P values by Kruskal-Wallis followed by Dunn multiple comparisons test). F, Summary graph of densitometry analysis of pPLN (Thr17) vs total PLN, normalized to GAPDH from control cardiomyocytes after β1AR or β2AR stimulation (n=5, P values by Kruskal-Wallis…

• 

  Figure 3.. L-type Ca2+ channels (LTCCs) changes between control and failing cardiomyocytes under local βAR stimulation. A, Left, representative traces of ICa,L currents before and after ISO application (1 µM) in the presence of PKA inhibitor (H89 10 µM). Right, summary I/V graph, inset showing the differences at −5 mV (n=5/3, P value by Wilcoxon matched-pairs signed rank test). B, Left, representative traces of ICa,L currents before and after ISO application (1 µM) in the presence of CaMKII (Ca2+/calmodulin-dependent protein kinase II) inhibitor (KN-93 10 µM). Right, Summary I/V graph, inset showing the differences at 0 mV (n=5/2, P value by Wilcoxon matched-pairs signed rank test). C, Representative traces of single LTCC recordings at −6.7 mV. D, Summary graph of the Po from control cardiomyocytes in TT and crest under local beta-2 adrenergic receptors (β1AR) or beta-2 adrenergic receptor (β2AR) stimulation (n=channels/cells/animals; 42/21/12, 28/15/9, 8/5/4, 21/10/8, 20/13/7, 13/5/2, *P<0.05,**P<0.01, by Kruskal-Wallis followed by Dunn multiple comparisons test). E, Summary graph of the chance of obtaining a LTCC current (% of occurrence). It represents the number of recordings with LTCC…

• 

  Figure 4.. Mechanism behind the local interaction between L-type Ca2+ channel (LTCC) and β2AR on the TT. A, Representative traces of single LTCC recordings at −6.7 mV. B, Summary graph of the Po from control cardiomyocytes after different preincubation treatments, for PKA inhibition (with H-89 10 µM or PKAi 3 µM), for CaMKII (Ca2+/calmodulin-dependent protein kinase II) inhibition (with KN-93 10 µM or AIP 5 µM), and for cholesterol removal (MβCD; n=channels/cells/animals; 42/21/12, 28/15/9, 10/7/4, 7/5/3, 8/6/4, 7/5/4, 8/5/5, 6/6/4, 10/5/2, 9/6/3, 7/4/2, 7/4/2; P values by Mann-Whitney U test). C, Representative WBs of control cardiomyocytes treated with MβCD with or without β2AR stimulation. D, Summary graph of densitometry analysis of pPLN normalized to α-actinin from control cardiomyocytes after MβCD with or without β2AR stimulation (n=4, P values by Mann-Whitney U test). E, Summary graph of densitometry analysis of CaMKII normalized to GAPDH from control cardiomyocytes after MβCD with or without β2AR stimulation (n=5, P values by Mann-Whitney U test). MβCD indicates methyl-β-cyclodextrin; and PKAi, inhibitory protein kinase peptide.

• 

  Figure 5.. REM1-265Cav blocks the local interaction between L-type Ca2+ channel (LTCC) and β2AR located in caveolae domains after 48 hours in culture. A, Representative single LTCC traces at −6.7 mV. B, Summary graph of the Po in TT and Crest from control cells, 48 hours culture cells with and without local β2AR stimulation, and 48-hour REM1-265Cav transfected cells with and without β2AR local stimulation (n=channels/cells/animals 42/21/12, 28/15/9, 11/8/4, 6/4/3, 4/3/3, 3/3/3, 21/10/8, 20/13/7, 7/6/3, 8/4/3, 5/4/4, 3/3/1, P values by Kruskal-Wallis followed by Dunn multiple comparisons test). C, Summary graph of the chance of obtaining a LTCC current (% of occurrence; P values by Fisher exact test).

• 

  Figure 6.. The local interaction between L-type Ca2+ channel (LTCC) and β2AR is lost in the tamoxifen-induced Cav3KO mouse model. A, Left, WB of control and Cav3KO mouse cardiomyocytes. Right, summary graph of densitometry analysis of cav3 normalized to GAPDH from control (n=6) and Cav3KO (n=6) cardiomyocytes (P value by Mann-Whitney U test). B, Left, 10×10 µm representative scanning ion conductance microscopy (SICM) images of control and Cav3KO cardiomyocytes. Right, summary graph showing the Z-groove index as a measure of surface regularity in control (n=99) and Cav3KO (n=52) cardiomyocytes (P value by Mann-Whitney U test). C, Representative traces of ICa,L currents in control and Cav3KO cardiomyocytes. D, I/V summary graph of ICa,L (Control n=14/4, Cav3KO n=16/4). E, ICa,L activation and inactivation curves in control and Cav3KO cardiomyocytes (Control n=14, Cav3KO n=16). F, Representative traces of single LTCC recordings at −6.7 mV. G, Summary graph of the Po in TT and Crest from control and Cav3KO cardiomyocytes, with and without local β2AR stimulation (n=channels/cells/animals 11/6/4, 13/6/3, 12/6/2, 11/5/3, 8/6/3, 7/5/3, 15/9/4, 10/6/3, *P<0.05, by Kruskal-Wallis followed by Dunn…

• 

  Figure 7.. Local β2AR stimulation can increase the Po of L-type Ca2+ channel (LTCC) in control human cardiomyocytes, but not in failing cardiomyocytes from patients with dilated cardiomyopathy (DCM). A, Representative traces of single LTCC recordings at −6.7 mV. B, Summary graph of the Po in TT and Crest from human control cardiomyocytes, with and without local β2AR stimulation (n=channels/cells/patients 9/4/3, 5/3/1, 7/2/1, 7/2/1, *P<0.05,***P<0.001 by Kruskal-Wallis followed by Dunn multiple comparisons test). C, Representation of the chance of obtaining a LTCC current (% of occurrence) in human control cardiomyocytes. D, Summary graph of the Po in TT and Crest from patients with DCM, with and without local β2AR stimulation (n=channels/cells/patients; 20/13/7, 10/5/2, 13/9/7, 6/5/2, *P<0.05, ***P<0.001 by Kruskal-Wallis followed by Dunn multiple comparisons test). E, Percentage of occurrence in failing cardiomyocytes from patients with DCM. F, I/V plots of single channel recordings from human control cardiomyocytes. G, Summary graph of the conductance analysis obtained from the slope in F (n=channels/cells/patients; 9/4/3, 5/3/1, 7/2/1, 7/2/1, *P<0.05, **P<0.01 by Kruskal-Wallis followed…

• 

  Figure 8.. Schematic representation of the proposed signaling mechanism between L-type Ca2+ channel (LTCC) and β2AR. A, Normal distribution of LTCC and βAR on the T-tubule and Crest microdomain. B, On failing cells, the loss of TTs redistributed LTCCs, increasing their presence on the crest. Multiple targets proteins are phosphorylated at baseline, due to the higher activity of PKA and CaMKII (Ca2+/calmodulin-dependent protein kinase II) as a compensatory mechanism for the reduction in function. Including LTCC on the crest and β2AR, which switch to the inhibitory Gi subunit. C, General catecholamine stimulation will preferably affect control cardiomyocytes via beta-1 adrenergic receptors (β1ARs), because of the higher expression than β2ARs. This will cause a global cytosolic increase of cAMP, and consequently a global activation of PKA and CaMKII. Increasing the activity of LTCCs. PKA will also phosphorylated β2AR, switching to Gi, working as negative feedback. D, On failing cells, a global stimulation will cause a small response, as PKA and CaMKII activity cannot be increased much further. Observed as a blunted response of LTCC activity. E, In healthy conditions, β2ARs signaling is…