CaMKII induces permeability transition through Drp1 phosphorylation during chronic β-AR stimulation

Abstract

Mitochondrial permeability transition pore (mPTP) is involved in cardiac dysfunction during chronic β-adrenergic receptor (β-AR) stimulation. The mechanism by which chronic β-AR stimulation leads to mPTP openings is elusive. Here, we show that chronic administration of isoproterenol (ISO) persistently increases the frequency of mPTP openings followed by mitochondrial damage and cardiac dysfunction. Mechanistically, this effect is mediated by phosphorylation of mitochondrial fission protein, dynamin-related protein 1 (Drp1), by Ca²⁺/calmodulin-dependent kinase II (CaMKII) at a serine 616 (S616) site. Mutating this phosphorylation site or inhibiting Drp1 activity blocks CaMKII- or ISO-induced mPTP opening and myocyte death in vitro and rescues heart hypertrophy in vivo. In human failing hearts, Drp1 phosphorylation at S616 is increased. These results uncover a pathway downstream of chronic β-AR stimulation that links CaMKII, Drp1 and mPTP to bridge cytosolic stress signal with mitochondrial dysfunction in the heart.

Figure 1. Chronic ISO stimulation persistently elevated mPTP openings.

(a) Representative images and traces of the onset of mitochondrial flashes (white boxes) colocalized with Δψ_m indicator TMRM in adult rat cardiomyocytes treated with or without ISO (100 nM for 12 h). Scale bars, 10 μm. (b) Time-course assay showing the effects of ISO treatment on mitochondrial flash frequency in adult cardiomyocytes. In Vehicle groups, N=27, 40, 31, and 58 cells from 3 to 6 rats in the time points of 10 min, 6, 12 and 18 h. In ISO groups, N=43, 26, 50 and 23 in the time points of 10 min, 6, 12 and 18 h. *P<0.05 versus Vehicle at the same time point. (c) ISO treatment increased flash frequency in a dose-dependent manner. N=31, 37, 18 and 12 cells from 3 to 6 rats in the groups of Vehicle, 100 nM, 1 μM and 10 μM ISO. *P<0.05 versus Vehicle group. (d) Cyclophilin D inhibitor, CsA, (1 μM, 30 min) abolished the increase of flash frequency induced by ISO (100 nM, 12 h). N=26, 13, 19 and 22 cells from 3 to 6 rats in the groups of Vehicle, CsA, ISO and CsA+ISO, respectively. *P<0.05 versus Vehicle group, ^#P<0.05 versus ISO group. (e) Knocking down mitochondrial Ca²⁺ uniporter (MCU) by short hairpin RNA (shMCU) blocked ISO-induced flashes. N=39, 14, 30 and 17 cells from 3 to 7 rats in the groups of Vehicle, shMCU, ISO and shMCU+ISO, respectively. *P<0.05 versus Vehicle group, ^#P<0.05 versus ISO group. (f) Pretreatment with mitochondrial antioxidant, mitoTEMPO (1 μM, 1 h) attenuated ISO-induced flashes. N=37, 11, 37 and 11 cells from 3 to 6 rats in the groups of Vehicle, mitoTEMPO, ISO and mitoTEMPO+ISO, respectively.*P<0.05 versus Vehicle group, ^#P<0.05 versus ISO group. Data in b–f are mean±s.e.m. The data were analysed using Student's t-test in b and One-way ANOVA followed by Turkey post-test in c–f.

Figure 2. Blocking ISO-induced mPTP openings prevented mitochondrial and myocyte dysfunction during chronic β-AR stimulation.

(a) Representative linescan confocal images showing the laser-induced permanent loss of Δψ_m in individual mitochondrion. CsA (1 μM) was added 12 h after ISO treatment, when the ISO-induced flash frequency reaches peak. (b) Quantification of the time from the start of scan to the sudden loss of Δψ_m. The shorter the time, the more sensitive the mitochondria to the laser. N=312, 383 and 274 mitochondria from 23 to 30 cells and three rats in the groups of Vehicle, ISO and ISO+CsA, respectively. (c) The intensity of TMRM fluorescence at the beginning of each scan was used to evaluate the basal Δψ_m in individual mitochondrion. (d) CsA reversed ISO-induced cellular oxidative stress reflected by the increased rate of DCF-DA fluorescence over 30 s (1 frame per second) (dF/dt). N=46, 53 and 22 cells from four rats in the groups of Vehicle, ISO and ISO+CsA, respectively. (e,f) CsA attenuated the reduction of Ca²⁺ transients (e) and cell contraction amplitude (f) after 48 h ISO treatment. N=21, 22 and 24 cells from three rats in the groups of Vehicle, ISO and ISO+CsA, respectively. (g) CsA rescued myocyte death after 48 h of ISO (1 μM) treatment measured by Trypan blue assay. N=903, 643 and 604 cells from four rats in the groups of Vehicle, ISO and ISO+CsA, respectively. (h) Knockout of CypD ameliorated cell death in cultured adult mouse cardiomycytes treated with ISO (100 nM, 48 h). N=637, 651, 565 and 693 cells from three mice in the groups of Vehicle, CypD KO, ISO and CypD KO+ISO, respectively. Data in b–h are mean±s.e.m. *P<0.05 versus Control group, ^#P<0.05 versus ISO group. The data were analysed using One-way ANOVA followed by Turkey post test in b–h.

Figure 3. ISO induced mPTP openings through β1-AR and CaMKII pathway.

(a) Pretreatment with β1-AR specific antagonist (CGP, 0.5 μM), but not β2-AR specific antagonist (ICI, 0.5 μM), blocked the ISO-induced flashes. N=38, 48, 36 and 31 cells from 3 to 5 rats in the groups of Vehicle, ISO, CGP+ISO and ICI+ISO, respectively. (b) Either the peptide inhibitor for PKA (PKI, 10 μM) or a cell-permeable inactive cAMP analogue (8-RP-cAMPs, 100 μM) failed to prevent the increase of flash after ISO treatment. N=25, 34, 20 and 19 cells from three rats in the groups of Vehicle, ISO, PKI+ISO and 8-RP+ISO, respectively. (c) CaMKII blockers, AIP (10 μM) and KN-93 (0.5 μM), inhibited ISO-induced flashes. KN-92, the inactive analogue of KN-93, was used as control. N=44, 55, 29, 30 and 31 cells from 3 to 5 rats in the groups of Vehicle, ISO, AIP+ISO, KN93+ISO and KN92+ISO, respectively. All the reagents in a–c were added to the myocytes 30 min before ISO treatment. (d) Inhibition of CaMKII activity by adenovirus-mediated overexpression of a dominant negative CaMKII (CaMKII DN) significantly attenuated mPTP openings 12 h after ISO treatment. N=20, 22, 18 and 20 cells from three rats in the groups of Vehicle, CaMKII DN, ISO and CaMKII DN+ISO, respectively. (e) CaMKII DN overexpression prolonged the time of laser-induced permanent loss of Δψ_m after ISO treatment (100 nM, 48 h). N=208, 179, 260 and 165 mitochondria from 15 to 25 cells and three rats in the groups of Vehicle, CaMKII DN, ISO and CaMKII DN+ISO, respectively. (f) CaMKII DN overexpression prevented ISO-induced cell death (1 μM, 48 h). N=474, 577 and 374 cells from four rats in the group of Vehicle, ISO and CaMKII DN+ISO, respectively. Data in a–f are mean±s.e.m. *P<0.05 versus Control group, ^#P<0.05 versus ISO group. The data were analysed using One-way ANOVA followed by Turkey post test in a–f.

Figure 4. Activating CaMKII pathway stimulated mPTP opening and mitochondrial stress in adult cardiomyocytes.

(a) Representative images of Western blot using the anti-HA antibody confirmed the expression of HA-tagged wild-type CaMKII (CaMKII WT) and a constitutively active CaMKII (CaMKII CA). (b) Overexpression of CaMKII WT or CaMKII CA significantly increased flash frequency in adult cardiomyocytes. N=26, 28 and 26 cells from four rats in the groups of control, CaMKII WT and CaMKII CA, respectively. *P<0.05 versus control group. (c) Inhibition of CaMKII activity by applying KN93 (0.5 μM) attenuated the increased flash frequency by CaMKII WT overexpression. N=7, 9 and 9 cells from three rats in the groups of Control, CaMKII WT and WT+KN93, respectively. *P<0.05 versus control group, ^#P<0.05 versus CaMKII WT group. (d) CsA (1 μM) inhibited the increased mPTP openings by CaMKII WT overexpression. N=14, 27 and 22 cells from three rats in the groups of control, CaMKII WT and WT+KN93, respectively. *P<0.05 versus Control group, ^#P<0.05 versus CaMKII WT group. (e) CaMKII WT overexpression promoted laser-induced permanent loss of Δψ_m in adult cardiomyocytes and showed additive effect with ISO treatment (100 nM, 12 h). N=536, 366, 305 and 242 mitochondria from 21 to 48 cells and four rats in the groups of control, ISO, CaMKII WT and WT+ISO, respectively. *P<0.05 versus Vehicle group, ^#P<0.05 versus CaMKII WT group. (f) CaMKII WT overexpression exaggerated cell death after ISO treatment (100 nM, 24 h). N=665, 577, 1,216 and 567 cell from five rats in the groups of Vehicle, CaMKII WT, ISO and WT+ISO, respectively. *P<0.05 versus ISO 24 h group. Data in b–f are mean±s.e.m. The data were analysed using One-way ANOVA followed by Turkey post test in b–f.

Figure 5. Chronic ISO infusion in vivo increased mPTP openings in the intact heart of mt-cpYFP transgenic mice and induced cardiac hypertrophy.

(a) Representative images and traces showing the onset of mitochondrial flashes (white boxes) accompanied by loss of Δψ_m (TMRM signal) in intact and Langendorff perfused heart. Scale bar, 10 μm. The mice were under ISO infusion (15 mg kg⁻¹ d⁻¹ for 2 weeks, ISO 2W) by a mini-osmotic pump implanted subcutaneously. (b) Summarized data showing the increase in flash frequency by ISO infusion and its blockade by KN93 (10 μM kg⁻¹ administered together with ISO). N=33, 27 and 25 serial scanning images from 4 to 6 mice in the groups of Vehicle, ISO and KN93+ISO, respectively. (c–e) ISO-induced cardiac hypertrophy was measured by heart weight to body weight ratio (c, N=5) and quantitative real-time PCR for ANP (d, N=4) and BNP (e, N=6). KN93 or β-blocker (Prop, 10 mg kg⁻¹) were administered together with ISO. Data in b–e are mean±s.e.m. *P<0.05 versus Vehicle group, ^#P<0.05 versus ISO group. The data were analysed using One-way ANOVA followed by Turkey post test in b–e.

Figure 6. Chronic ISO administration promoted Drp1 S616 phosphorylation and its mitochondrial translocation through CaMKII pathway.

(a) Increased Drp1 phosphorylation at S616 site (Drp1^S616) in the heart after ISO infusion through CaMKII-dependent pathway. N=5, 6, 4 and 4 mice in the groups of Vehicle, ISO, Prop+ISO and KN93+ISO, respectively. (b) ISO administration induced Drp1^S616 phosphorylation in cultured adult rat cardiomyocytes. N=6. (c) CaMKII blocker, KN-93 (0.5 μM), prevented Drp1^S616 phosphorylation by ISO (100 nM for 12 h). N=4. (d) CaMKII WT overexpression induced Drp1^S616 phosphorylation in adult cardiomycytes. N=4. (e) Representative immunoblots and quantification of Drp1 proteins in mitochondrial and cytosolic fractions of the heart after ISO infusion. COX IV and β-actin were used as mitochondrial and cytosolic markers, respectively. N=5, 6 and 4 mice in the groups of Vehicle, ISO and KN93+ISO, respectively. (f) Immunofluorescent analysis showing increased ‘punctate' Drp1 staining colocalized with mitochondria. Images are representative of 30 cells from three rats in each group. (g) Representative immunoblots and quantification of Drp1^S616 or Drp1^S637 phosphorylation in the mitochondrial or cytosolic fractions of the heart after 2-weeks of ISO infusion. N=4. (h) ISO treatment (1 μM or 10 μM for 24 h) induced mitochondrial fragmentation in H9C2 cardiac myoblasts. A dominant negative Drp1 mutation (Drp1 K38A) was used as positive control. Form factor (FF; the reciprocal of circularity value) and aspect ratio (AR; major axis/minor axis) were acquired by using ImageJ. Smaller values of AR and FF indicate increased mitochondrial fragmentation. N=17,714, 5,929 and 5,862 mitochondria in the groups of Vehicle, ISO 1 μM and ISO 10 μM, respectively. (i) Overexpression of CaMKII WT and CaMKII CA increased mitochondrial fragmentation in H9C2 cells. CaMKII WT potentiated the effects of ISO on mitochondrial morphological change as indicated by significant fragmentation at a low ISO dose (100 nM). N=17,714, 17,692, 9,567 and 17,035 mitochondria in the groups of Vehicle, CaMKII WT, WT+ISO and CaMKII CA, respectively. Data are mean±s.e.m. *P<0.05 versus Vehicle, ^#P<0.05 versus ISO. The data were analysed using One-way ANOVA followed by Turkey post test.

Figure 7. CaMKII binds and directly phosphorylates Drp1 at S616.

(a) Co-immunoprecipitation analysis showing the binding of Drp1 with CaMKII in adult cardiomyocytes. Images are representative of four repeats. (b) HA-tagged CaMKII from H9C2 cells were attached to anti-HA magnetic beads and incubated with WT Drp1 or S616A mutation purified from E. coli. (2 μM) in the presence or absence of calmodulin (1 mM) and Ca²⁺ (1 mM) for 24 h. The supernatant was used for Western blot to detect Drp1 S616 phosphorylation. Images are representative of three repeats. (c) The beads were boiled and samples were used for Western blot using anti-HA (for CaMKII) or anti-Drp1 antibodies for determining the in vitro interaction between CaMKII and Drp1. Images are representative of three repeats.

Figure 8. Drp1 inhibition suppressed ISO-induced mitochondrial and myocyte dysfunction and heart hypertrophy.

(a) Preventing Drp1^S616 phosphorylation by overexpression a phosphorylation null mutation of Drp1 (Drp1 S616A, in which serine at 616 was mutated to alanine) blocked ISO-induced flash frequency in adult cardiomyocytes. N=12, 14, 13 and 10 cells from three rats in the groups of Vehicle, Drp1 S616A, ISO and S616A+ISO, respectively. (b) Drp1 S616A also prolonged the time of laser-induced permanent loss of Δψ_m by chronic ISO treatment (1 μM, 48 h). N=193, 245, 231 and 224 mitochondria from 20 to 24 cells from three rats in the groups of Vehicle, Drp1 S616A, ISO and S616A+ISO, respectively. (c) Drp1 S616A rescued myocyte death induced by ISO treatment (10 μM, 48 h). N=1061, 985, 877 and 694 cells from three rats in the groups of Vehicle, Drp1 S616A, ISO and S616A+ISO, respectively. (d–f) Drp1 K38A also blocked ISO's effect on flash frequency (d), laser-induced permanent loss of Δψ_m (e) and myocyte death (f). In d, N=26, 11, 36, 17 cells; in e, N=479, 239, 198 and 196 mitochondria; and in f, N=536, 653, 697 and 819 cells in the groups of Vehicle, Drp1 K38A, ISO and K38A+ISO, respectively. (g) Mdivi-1 (50 mg kg⁻¹ d⁻¹), a chemical inhibitor of Drp1, efficiently attenuated the increased flash frequency in intact heart by 2 weeks of ISO infusion. N=25, 18 and 27 images from 4 to 6 hearts in the groups of Vehicle, ISO and Mdivi-1+ISO, respectively. (h) Mdivi-1 prevented ISO-induced cardiac hypertrophy. N=7–8 mice. Data in a–h are mean±s.e.m. *P<0.05 versus Vehicle group, ^#P<0.05 versus ISO group. (i) Representative images and summarized data showing Drp1^S616 phosphorylation and total Drp1 levels in the ventricular samples of heart failure patients. N=4 for each group. *P<0.05 versus None-failing control group. Data are mean±s.e.m. In a–i, the data were analysed using One-way ANOVA followed by Turkey post test.

Figure 9. Schematic model of CaMKII mediating mPTP through Drp1^S616 phosphorylation during chronic β-AR stimulation.

Sustained ISO treatment activates CaMKII pathway, a downstream kinase of β1-AR, and subsequently increases the phosphorylation of Drp1 at S616 (Drp1^S616), which activates Drp1. After translocating to the outer membrane of mitochondria, the phosphorylated Drp1 triggers fission and mPTP openings, which are recorded by mitochondrial flash events. Finally, chronic activation of this pathway leads to mitochondrial and myocyte dysfunction. Abolishing CaMKII activity (CaMKII DN, KN93 or AIP), inhibiting Drp1 activity (Drp1 K38A or Mdivi-1), preventing Drp1^S616 phosphorylation (Drp1 S616A), or blocking mPTP openings (CsA or CypD KO) efficiently prevented myocyte damage and cardiac hypertrophy during chronic β1-AR stimulation.