Perinuclear damage from nuclear envelope deterioration elicits stress responses that contribute to LMNA cardiomyopathy

Abstract

Mutations in the LMNA gene encoding lamins A/C cause an array of tissue-selective diseases, with the heart being the most commonly affected organ. Despite progress in understanding the perturbations emanating from LMNA mutations, an integrative understanding of the pathogenesis underlying cardiac dysfunction remains elusive. Using a novel conditional deletion model capable of translatome profiling, we observed that cardiomyocyte-specific Lmna deletion in adult mice led to rapid cardiomyopathy with pathological remodeling. Before cardiac dysfunction, Lmna-deleted cardiomyocytes displayed nuclear abnormalities, Golgi dilation/fragmentation, and CREB3-mediated stress activation. Translatome profiling identified MED25 activation, a transcriptional cofactor that regulates Golgi stress. Autophagy is disrupted in the hearts of these mice, which can be recapitulated by disrupting the Golgi. Systemic administration of modulators of autophagy or ER stress significantly delayed cardiac dysfunction and prolonged survival. These studies support a hypothesis wherein stress responses emanating from the perinuclear space contribute to the LMNA cardiomyopathy development.

Fig. 1.. Rapid cardiac performance deterioration after CM-specific Lmna deletion.

(A) Tam dosing schedule. Red arrows denote days of Tam injection and blue arrows show 2 days of rest. “12w old” denotes 12-week-old mice. (B) Hearts excised from CM-CreTRAP:Lmna^flox/flox mice treated with vehicle (Veh) or Tam for 2 and 4 weeks. (C) Echo on CM-CreTRAP:Lmna^flox/+ (diamond) and CM-CreTRAP:Lmna^flox/flox (circle) at 2 and 4 weeks Tam treatment. *P = 0.015 and **P < 0.0001 using one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison correction with the Veh group serving as comparison control. Error bars = SEM. Blue = male and red = female mice. (D) Tabulation of echo data ± SEM. FS, fractional shortening; EF, ejection fraction; LVESD, left ventricular end systolic dimension; LVEDD, left ventricular end diastolic dimension; LVESPW, left ventricular end systolic posterior wall; LVEDPW, left ventricular end diastolic posterior wall. Veh-treated mice at 2 and 4 weeks were measured together with the Tam-treated group but pooled and presented as a single Veh control group because no differences were noted between them.

Fig. 2.. Pathological cardiac remodeling accompanies cardiac dysfunction.

(A) Histological analyses of the hearts from CM-CreTRAP:Lmna^flox/flox mice at 1, 2, and 4 weeks after Tam or Veh treatment. Blue, yellow, and green arrows denote degenerating myocytes, pyknotic nuclei, and vacuolation, respectively. Black arrows denote insets. H&E, hematoxylin and eosin; WGA, wheat germ agglutinin. Scale bars, 50 μm. A representative image per group (n = 3) are shown. (B) Quantitation of average % fibrosis of Masson’s trichrome staining from 12 independent images from three mouse hearts per group. Error bars = SEM. % Fibrosis P value was determined using one-way ANOVA and Dunnett’s correction with Veh as control. (C) Quantitation of myocyte cross-sectional area based on WGA staining. Three images were taken per section, and average area of 30 individual CMs was measured in total per group across three experiments. Error bars = SD. P values were determined using one-way ANOVA and Dunnett’s correction with Veh as control. (D) qPCR of cardiac stress and profibrotic markers in hearts of Tam-treated CM-CreTRAP:Lmna^flox/flox mice. 1w to 4w denote weeks after final Tam dosing. Data are presented as fold change relative to Veh. *P < 0.05, **P <0.01, and ***P < 0.0001 using one-way ANOVA with Dunnett’s correction with Veh as control. Error bars = SEM. n = 3. (E) Immunofluorescence images of platelet-derived growth factor receptor α (PDGFRα), vimentin, α–smooth muscle actin (αSMA), EGFP-L10a, and desmin on mouse hearts 4 weeks after Veh or Tam treatment. The red and green numbers in line with the PDGFRα and vimentin micrographs denote their quantitation (% area per field ± SEM), respectively, calculated from 15 images from three mice per group across three replicate experiments. White boxes denote magnified insets shown to the right. Scale bars, 50 μm. Data from both sexes are shown in this figure.

Fig. 3.. Selective UPR activation in Lmna-deleted hearts.

(A) H&E staining of longitudinal heart sections from CM-CreTRAP:Lmna^flox/flox mice 2 weeks after Veh or Tam. Yellow arrows highlight abnormally shaped, ruptured, and stretched nuclei with green arrows showing inset. Average number ± SEM of abnormal nuclei per field counted from 15 fields from three mice per group from three experiments are shown. (B) TEM on hearts from CM-CreTRAP:Lmna^flox/flox mice 2 weeks after Veh or Tam. Red boxes show magnified perinuclear space. Yellow and black arrowheads denote Golgi and 100 nm vesicles, respectively. m, mitochondria; n, nucleus. Scale bars, 1 μm. Numbers in line with EM micrographs denote percent abnormal nuclei quantified in Veh (n = 45 total nuclei) and 2 weeks after Tam (n = 31) from two independent hearts per group. (C) Immunoblot of lamin A/C, PERK, ATF4, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in hearts from Veh or Tam-treated CM-CreTRAP:Lmna^flox/flox mice. Numbers on top denote individual hearts. One and 2 weeks denote time after final Tam treatment. Numbers in line with blots show quantification of protein bands normalized to GAPDH in arbitrary units. (D) qPCR analyses of mediators of unfolded protein response in hearts from CM-CreTRAP:Lmna^flox/flox mice treated with Veh or Tam. 1w, 2w, and 4w denote weeks after Tam. *P <0.05, **P <0.005, and #P <0.0001 using one-way ANOVA with Dunnett’s’s post hoc with Veh as control. n = 5 except for 1w (n = 3). All error bars in this figure = SEM. (E) Schema of experimental design using TRAP. Right panel shows qPCR on CM-specific translating mRNAs probed for Eif2ak3, Atf4, Ddit3, and Hspa5, respectively, from CM-CreTRAP:Lmna^flox/flox mice 1 to 4 weeks after Tam. *P < 0.05 and **P < 0.0005 using one-way ANOVA with Dunnett’s’s post hoc with Veh as control. n = 3.

Fig. 4.. Golgi stress in Lmna-deleted CMs.

(A) TEM of Lmna-deleted mouse hearts. Dotted boxes denotes inset. Yellow brackets denotes Golgi. Blue/red arrowheads denote outer/inner nuclear membranes. Scale bars, 200 nm. Quantitation of % dilated and normal Golgi in Veh (n = 45 nuclei), 2 weeks (n = 31), and 4 weeks (n = 27) after Tam. (B) CREB3/GAPDH immunoblots (left) and qPCR of CREB3-activated genes (right) in monensin-treated Lmna^flox/flox MEFs. Arrowheads denotes CREB3 cleavage. n = 3. (C) RT-PCR of Xbp1 mRNA splicing in monensin (2 μM)– or BFA (20 μM)–treated Lmna^flox/flox MEFs. Xbp1u and Xbp1s denote unspliced and spliced variants. Numbers on bottom denote Gapdh Ct values (input control). Representative image from three experiments are shown. (D) qPCR of UPR mediators in Lmna^flox/flox MEFs treated as in (C). n = 3. NS, not significant. (E) Representative CREB3 and DAPI images of AdBlank or AdCre-infected Lmna^flox/flox MEFs from three experiments. White lines denote linear fluorescence intensity measurement examples. Scale bar, 50 μm. (F) Fluorescence intensity profiles of CREB3 in AdBlank or AdCre-infected Lmna^flox/flox MEFs. Measurement directionality is from cytoplasm (cyto) to nucleus (nucl). (G) CREB3, LMNA, and GAPDH immunoblots in AdBlank (bl)– or AdCre (cre)–infected Lmna^flox/flox MEFs. Black and gray arrowheads denote CREB3 cleavage and lamin A/C, respectively. Asterisk denotes nonspecific band. Representative blot from n = 3 experiments. (H) Representative CREB3 and GAPDH immunoblot of adult CMs from Veh- or Tam-treated CM-CreTRAP:Lmna^flox/flox mice. n = 3 experiments. Numbers on top denote individual hearts. Arrowheads denote CREB3 and its fragments. Remaining graphs show qPCR on TRAP mRNA for Arf4, Trappc13, Tnfrsf1b, and Tnfrsf10b. n = 3 to 5. (I) Confocal CREB3 and DAPI images on adult CMs from Veh- or Tam-treated CM-CreTRAP:Lmna^flox/flox mice from three isolation per staining. Scale bar, 10 μm. (J) CREB3 quantification from (I). n = nuclei analyzed from three experiments. All error bars in this figure = SEM. All P = one-way ANOVA with Dunnett’s post hoc with *P < 0.05, **P < 0.005, and ***P < 0.0005.

Fig. 5.. MED25 depletion sensitizes cells to Golgi stress–induced vacuolar cell death.

(A) Experimental schema of TRAP/RNA-seq. (B) Volcano plot of differentially expressed genes sorted by –log₁₀ of nominal P value with 5% FDR cutoff. (C) Heatmap of top 30 coding genes filtered by adjusted P values of perfect markers. Red arrows denote the position of Med6 and Med25. (D) Viability of MED25-depleted C2C12 treated with monensin, 20 μM PF-429242 (PF), and 20 μM PF-429242 + 2 μM monensin. The % cell viability was calculated relative to Veh controls. P values were calculated using two-way ANOVA with Dunnett’s correction. Error bars = SEM. n = 3 experiments. (E) Representative phase contrast images of blank control (ctrl) and MED25 KD C2C12 cells treated with Veh, monensin, PF-429242, and cotreatment. Insets, denoted by red boxes, show magnified cells with or without vacuoles. Scale bars, 50 μm. (F) CREB3 and GAPDH immunoblot of C2C12 (left) and 3T3-L1 (right) with MED25 KD (sh1 and sh2) treated with 2 μM monensin for 3 and 24 hours. Arrowheads denote CREB3 cleavage. Numbers in line with blots denote cleaved CREB3 quantitation normalized to GAPDH in arbitrary units. Bottom panels show cleaved CREB3 presented as fold change relative to ctrl at 0 hours (set to 1) from three experiments. P values were calculated using one-way ANOVA with Dunnett’s correction. (G) Viability of MED25-depleted C2C12 cells treated for 24 hours with chemical inducers of Golgi (monensin, nigericin, and golgicide A) and ER (BFA, thapsigargin, and tunicamycin) stress. The % cell viability was calculated relative to their respective cells treated with Veh. P values were calculated using two-way ANOVA with Dunnett’s correction. Error bars = SEM. n = 3. ns = not significant.

Fig. 6.. Perinuclear damage underlies accumulation of autophagic markers.

(A) Immunoblot of p62, lipidated LC3B[LC3B-II], and GAPDH on Veh- or Tam-treated CM-CreTRAP:Lmna^flox/flox hearts at indicated times since last Tam dosing. n = 3 hearts per group. Numbers below blots denote quantified bands normalized to GAPDH in arbitrary units. (B) Compiled quantitation of LC3B-II and p62 blots in (A). (C) Representative immunofluorescence of C2C12 with Control or Gorasp1/Gorasp2 DKD stained for RCAS1 and p62 from three experiments. Control denotes C2C12 infected with lentivirus generated from blank plasmid. Scale bar, 20 μm. (D) Immunoblot of C2C12 with Gorasp1 or Gorasp2 KD alone or Gorasp1/Gorasp2 DKD probed for p62 and LC3B-II. GAPDH was assessed as loading control. Numbers below blots denote p62 and LC3B-II quantitation normalized to GAPDH in arbitrary units. Bottom shows LC3B-II and p62 fold change values relative to ctrl (set to 1) from n = 3 experiments. (E) Immunoblot/quantitation of monensin-treated C2C12 nuclear extracts with Gorasp1/2 KD probed for MED25 and β-actin as in (D). (F) Immunofluorescence of Control and Gorasp1/Gorasp2 DKD C2C12 transduced with EGFP-LC3B encoding retroviruses. After 1-week recovery, cells were stained for p62 and DAPI. Insets show magnified images of EGFP-LC3B puncta with p62. Numbers in line denote Mander’s coefficient ± SD (fraction of p62 overlapping with LC3B) from 36 random data points per group from three experiments. Scale bars, 10 μm. (G) Representative immunofluorescence images of Lmna^flox/flox MEFs 5 days after infection with AdBlank or AdCre stained for RCAS1 and DAPI from three experiments. Yellow arrowheads denote ruptured nuclei with extruding DNA. White arrow denotes nucleus with the highest nuclear irregularity index (NII). Higher NII denotes increased irregularities. Red arrow denotes nucleus with minor herniation but largely intact nuclear shape and Golgi. Scale bar, 20 μm. (H) NII plotted against perinuclear RCAS1 signal area from Lmna^flox/flox MEFs infected with AdBlank or AdCre. Data points were compiled from three experiments.

Fig. 7.. Modulating autophagy or ER stress attenuates cardiomyopathy development.

(A) Schema of therapy schedule. (B) Echo on CM-CreTRAP:Lmna^flox/flox mice at 2 and 4 weeks after Tam, Tam + metformin (T/M), or Tam + azoramide (T/A). LVESD and LVEDD denote left ventricular end systolic and diastolic diameters, respectively. P values were obtained using one-way ANOVA with Tukey’s correction. Error bars = SEM. (C) Representative H&E and Masson’s Trichrome staining of heart sections from CM-CreTRAP:Lmna^flox/flox mice at 4 weeks after treatments described in (A) from three mice per group. Scale bars, 100 μm. (D) Heatmap of mRNA expression profile on natriuretic peptide precursors (red), profibrotic (blue), ER stress–related (purple), autophagy (black), and Golgi stress (green)–related genes in hearts from CM-CreTRAP:Lmna^flox/flox mice at 4 weeks after treatments described in (A). Numbers on top of heatmap denote biological replicates. Color gradient scales and values on bottom denote fold change over mean value of 4w Tam from the five biological replicates. P values were obtained using unpaired, two-tailed Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001. (E) p62, LC3B, and GAPDH immunoblots on CM-CreTRAP:Lmna^flox/flox mice hearts at 4 weeks after Veh, Tam, and Tam + metformin. Numbers on top denote biological replicates. Numbers below blots denote band quantitation normalized to GAPDH in arbitrary units. Bottom shows compiled quantitation from biological replicates. P values were obtained using one-way ANOVA with Tukey’s correction. *P < 0.05, **P < 0.01, and ***P < 0.001. Error bars = SEM. (F) Immunoblot similar to above but with azoramide therapy and probed for CHOP, phospho-eIF2α (peIF2α), and eIF2α. (G) Kaplan-Meier plot of CreTRAP:Lmna^flox/flox mice treated with Tam (n = 8), Tam + metformin (n = 10), and Tam + azoramide (n = 9). Tam-treated CreTRAP:Lmna^flox/+ (n = 5) and Veh-treated CreTRAP:Lmna^flox/flox (n = 5) mice are presented as a single group. P values were calculated using log-rank test with the survival curve of CreTRAP:Lmna^flox/flox mice + Tam as the comparison control. Sex of mice at death is shown within the curve. WB, Western blot.