RGS11-CaMKII complex mediated redox control attenuates chemotherapy-induced cardiac fibrosis

Fig. 1. – RGS11 is downregulated in the human heart following chemotherapy exposure. We collected post-mortem heart tissue samples from individuals with a history of chemotherapeutic treatment with regimens containing 5-FU or an anthracycline.(A) Representative cardiac staining for fibrosis (Masson Trichrome) and immunohistochemistry of troponin T or (B) RGS11 in a control or chemotherapy exposed patients (n = 10) [scale bar = 100 μm]. (C) Correlation between RGS11 histoscores and fibrosis or cardiac troponin levels in control or chemotherapy exposed patients (n = 10). (D) Samples from chemotherapy-exposed patients were stratified based on cardiac health and classified into 2 groups: no heart phenotype and heart phenotype with fibrosis. Protein immunoreactivity was determined via western blotting in cardiac tissue (n = 4–10). β-Actin serves as a loading control for western blots. Immunoblots are accompanied by a densitometric quantification wherein expression is normalized to the corresponding control group. (E) Representative cardiac staining for 4HNE with quantification (n = 10) [scale bar = 100 μm]. Data were analyzed by student's t-test or one- or two-way ANOVA with Sidak's post-hoc…

Fig. 2. RGS11 prevents doxorubicin-induced oxidative stress, mitochondrial dysfunction, and cell death in human cardiomyocytes. (A) Human AC-16 cardiomyocytes were treated with doxorubicin (3 μM, 16 h), 5-FU (500 μM, 16 h), or oxaliplatin (0.06 mM, 16 h) and immunoblotting performed probing for RSG11 (n = 3). (B–H) Control (scramble plasmid) or RGS11 OE AC-16 cells were treated with doxorubicin (3 μM, 16 h) ± pre-treatment with mitochondrial calcium uniporter inhibitor Ru360 (50 μM, 1 h) or mitochondrial permeability transition pore (mPTP) blocker cyclosporin A (0.2 mM, 45 min) where indicated. (B) Immunoblotting for RGS11 (n = 6).(C) GPX activity (n = 5) and SOD activity (n = 5). (D) Mitochondrial Ca2+ flux (n = 5). (E) Mitochondrial membrane potential (ΔψM; n = 5). (F) Caspase-3 cleavage (n = 5). (G) Apoptosis (cytoplasmic histone-associated DNA fragments; n = 5). (H) Annexin V+ cells (n = 5). β-Actin serves as a loading control for western blots. Immunoblots are accompanied by a densitometric quantification wherein expression is normalized to the corresponding control group. Data were analyzed by one-way ANOVA with Sidak's post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P <…

Fig. 3. RGS11 forms a complex with CaMKII and blocks CaMKII-dependent cardiomyocyte damage. (A) Representative cardiac staining for CaMKII in a control or chemotherapy exposed patients [n = 10, scale bar = 100 μm]. (B) Correlation between CaMKII histoscores andfibrosis or cardiac troponin levels in control or chemotherapy exposed patients (n = 10). (C) Immunoblotting for phosphorylated and oxidized CaMKII protein expression in heart tissue from chemotherapy exposed patients or matched controls (n = 4–5). (D) Reciprocal co-immunoprecipitation of RGS11 and CaMKII from AC-16 cells. (E) Co-immunoprecipitation of CaMKII with RGS11 deletion constructs. (F) In silico modeling of the putative RGS11-CaMKII complex revealed key RGS11 residues predicted to support a direct interaction between RGS11 and CaMKII. (G) Control or RGS11 CRISPR KO AC-16 cells were treated with CaMKII inhibitor KN-93 (50 μM, 1 h). CM-H2-DCFDA fluorescence (total ROS), cell viability, apoptosis (cytoplasmic histone-associated DNA fragments; n = 5) were measured. (H) RGS11 KO AC-16 cells were transfected with control plasmid (vector) ± phosphorylation (T287A) or oxidation (M281/282 Vhourss)) deficient CaMKII (HA-tagged)…

Fig. 4. RGS11 regulates ATF3 in cardiomyocytes. (A) Representative cardiac staining for ATF3 in a control or chemotherapy exposed patients [n = 10, scale bar = 100 μm] and correlation between ATF3 histoscores and fibrosis or cardiac troponin levels in control or chemotherapy exposed patients (n = 10). (B) Immunoblotting for ATF3 and NRG1 protein expression in heart tissue from chemotherapy exposed patients with or without fibrosis or matched controls (n = 4–5). (C–E) Control or RGS11 CRISPR KO AC-16 cells were treated with doxorubicin (3 μM, 16 h) ± ATF3 shRNA for 12 h. (C) CM-H2-DCFDA fluorescence (total ROS; n = 5). (D) Apoptosis (cytoplasmic histone-associated DNA fragments; n = 5). (E) Protein immunoreactivity was determined via western blotting (n = 6). (F–I) Control or RGS11 KO AC-16 cells were treated with doxorubicin (3 μM, 16 h) ± pre-treatment with ATF3 shRNA for 12 h or Nox blocker DPI (1 μM) or pan-ErbB blocker cI-1033 (2 μM). (F) CM-H2-DCFDA fluorescence (total ROS; n = 5). (G–H) NRG1 concentration in cell culture media (n = 5). (I) Apoptosis (cytoplasmic histone-associated DNA fragments; n = 5). β-Actin serves as a loading control for western blots. Immunoblots are…

Fig. 5. RGS11-dependent regulation of ATF3 requires CaMKII. (A) Co-immunoprecipitation of ATF3 with RGS11or CaMKII in AC-16 cells. (B) Control or RGS11 (n = 3) CRISPR KO AC-16 cells were transfected with control plasmid (vector) ± CaMKII (HA-tagged) or phosphorylation (T287A) or oxidation (M281/282 V) deficient CaMKII (HA-tagged) where indicated. (C–E) Control (scramble shRNA) or ATF3 KD VCM were treated with doxorubicin (3 μM, 16 h) ± pre-treatment with CaMKII inhibitor KN-93 (50 μM, 1 h). (C) NRG1 concentration in cell culture media (n = 5). (D) CM-H2-DCFDA fluorescence (total ROS; n = 5). (E) Apoptosis (cytoplasmic histone-associated DNA fragments; n = 5). Immunoblots are accompanied by a densitometric quantification wherein expression is normalized to the corresponding control group. β-Actin serves as a loading control for western blots. Data were analyzed by student's t-test or one- or two-way ANOVA with Sidak's post-hoc test. *P < 0.05, **P < 0.01, ****P < 0.0001. ns = not significant. Data are presented as mean ± SEM.

Fig. 6. RGS11/CaMKII regulate EC/VCM crosstalk. (A) RGS11 expression (immunoblotting) in heart tissue, VCM, VCF, and EC. (B) Isolated mice ECor VCM were treated with doxorubicin (3 μM, 12 h), culture media was removed and added to VCM and EC, respectively. Immunoblotting was performed to detect RGS11 (n = 3). (C) HUVEC cells were treated with doxorubicin (3 μM, 16 hVV). Culture media was removed and added to control or RGS11 OE AC-16 cells. Immunoblotting was performed to detect RGS11, p-CaMKII, ox-CaMKII, iNOS, and NRG1 (n = 3). (D) Control or RGS11 OE HUVEC cells were treated with doxorubicin (3 μM, 16 h). Culture media was removed and added to AC-16 cultures. Immunoblotting was performed to detect RGS11, p-CaMKII, and ox-CaMKII (n = 3). (E) Control, RGS11 OE HUVEC cells were treated with doxorubicin (3 μM, 16 h). Immunoblotting was performed to detect NRG1 (n = 3). (F–G) HUVEC cells were treated with doxorubicin (3 μM, 16 h) and the culture media was transplanted on AC-16 cell cultures ± pre-treatment with pan-ErbB blocker cI-1033 (2 μM, 1 h). (F) CM-H2-DCFDA fluorescence (total ROS; n = 5). (G) Apoptosis (cytoplasmic histone-associated DNA fragments; n = 5). β-Actin serves as a…

Fig. 7. Cardiac-specific RGS11 OE protects against doxorubicin cardiotoxicity in mice. (A) Mice were treated with doxorubicin (cumulative dose of 45 mg/kg i. p.) or saline control over 8 weeks (chronic) or mice were given a single dose of doxorubicin (20 mg/kg i. p.) or saline and sacrificed after 3 days (acute). Cardiac tissues were collected from both the sets for protein expression analyses of RGS11, p-CaMKII, ox-CaMKII, CaMKII and ATF3 (n = 8). (B–G) An RGS11 encoding viral construct or control (Ad-β-gal) was introduced into the myocardium. After 15 days, animals were given saline or doxorubicin (cumulative dose of 45 mg/kg i. p.) over 8 weeks. After 8 weeks cardiac phenotyping was performed, and tissues samples collected 1 week later for biochemical and histological analyses. (B) Representative images [scale bar = 100 μm] of RGS11 and CaMKII immunohistochemistry and cardiac fibrosis (Masson Trichrome staining). Quantification of (C) RGS11 and (D) CaMKII histoscores (n = 10). (E) Quantification of collagen deposition (fibrosis, n = 10). (F) Average myocyte cross sectional area (n = 10). (G) CM-H2-DCFDA fluorescence (total ROS; n = 5). (H) TUNEL positive cells (n = 5). (I) Cardiac…

Fig. 8. RGS11 KD in heart triggers CaMKII-dependent cardiac fibrosis. A viral construct encoding RGS11 shRNA or control (scramble) was introduced into the myocardium. After 15 days, animals were given saline or KN-93 (8 mg/kgi. p, 2 doses, 4 days apart) and tissues collected 2 days after the last injection for downstream analyses. (A) Immunostaining for RGS11 in cardiac tissue. (B) Representative images [scale bar = 100 μm] and quantification of cardiac fibrosis (Masson Trichrome staining; n = 10). (C) Immunoblotting for RGS11, p-CaMKII, ox-CaMKII, ATF3, NRG1, αSMA and ANP in cardiac tissue (n = 3). (D) A viral construct encoding RGS11 shRNA or control (scramble) was introduced into the myocardium. After 15 days, animals were given saline or doxorubicin (cumulative dose of 45 mg/kg i. p.) and tissues collected after 9 weeks for histological analyses. Representative images depicting RGS11 staining, CaMKII staining, Masson Trichrome staining (fibrosis), and H&E staining (myofibrillar architecture) in doxorubicin treated control and cardiac RGS11-KD mice [scale bar = 100 μm] and quantification. (E) Schematic depicting the role of RGS11 in regulation of CaMKII/ATF3 in cardiac myocytes and…