SIRT1 Ameliorates Lamin A/C Deficiency-Induced Cardiac Dysfunction by Promoting Mitochondrial Bioenergetics

Abstract

Dilated cardiomyopathy (DCM) is associated with high mortality despite advanced therapies. The LMNA gene encodes lamin A/C and is the second most frequently mutated gene associated with DCM, for which therapeutic options are limited. Here we generated Lmna ^-/- mice and found they exhibited cardiac dysfunction at the age of 1 month but not at 2 weeks. Proteomics showed down-regulation of mitochondrial function-related pathways in Lmna ^-/- hearts. Moreover, early injured mitochondria with decreased cristae density and sirtuin 1 (SIRT1) down-regulation were observed in 2-week-old Lmna ^-/- hearts. Adenoviral overexpression of SIRT1 in lamin A/C knockdown neonatal rat ventricular myocytes improved mitochondrial oxidative respiration capacity. Adeno-associated virus-mediated SIRT1 overexpression alleviated mitochondrial injury, cardiac systolic dysfunction, ventricular dilation, and fibrosis, and prolonged lifespan in Lmna ^-/- mice. Mechanistically, LMNA maintains mitochondrial bioenergetics through the SIRT1-PARKIN axis. Our results suggest that targeting the SIRT1 signaling pathway is expected to be a novel therapeutic strategy for LMNA mutation-associated DCM.

Keywords: LMNA; SIRT1; dilated cardiomyopathy; mitochondrial bioenergetics; proteomics.

Graphical abstract

Figure 1

One-Month-Old Lmna^–/– Mice Exhibit Cardiac Dysfunction (A) Representative exteriors (scale bar: 2 cm) and cardiac appearances (scale bar: 2 mm) of 1-month-old wild-type (WT) and Lmna^–/– mice. (B) Heart weight (HW)/body weight (BW) (n = 7) and lung weight (LW)/BW (WT, n = 5; Lmna^–/–, n = 4) of WT and Lmna^–/– mice. (C) Representative echocardiography of WT and Lmna^–/– mice. (D) Left ventricular ejection fraction (LVEF), fractional shortening (FS), and heart rate (HR) in WT and Lmna^–/– mice (n = 7). (E) Left ventricular end-systolic volume (LVESV)/BW, left ventricular end-diastolic volume (LVEDV)/BW, left ventricular end-systolic diameter (LVIDs)/BW, and left ventricular end-diastolic diameter (LVIDd)/BW in WT and Lmna^–/– mice (n = 7). (F) Representative hematoxylin and eosin (HE) staining (scale bar: 1 mm) and Masson’s trichrome staining (scale bar: 100 μm) images of the ventricular transverse section of WT and Lmna^–/– mice. (G) Quantification of the percentage of collagen in myocardial tissue of WT and Lmna^–/– mice (n = 5). (H) Kaplan-Meier curves of WT and Lmna^–/– mice showing a median survival time of 36 days for Lmna^–/– mice (n = 8, Breslow generalized Wilcoxon test). Data are presented as mean ± SD. Data in B, D, E (LVIDs/BW, LVIDd/BW), and G were compared by using Student’s t-test, and data in E (LVESV/BW, LVEDV/BW) were compared by using the Mann-Whitney U test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 2

Enrichment Analysis of DEPs Between WT and Lmna^–/– Mice Hearts (A) Principal component analysis (PCA) of proteins in 1-month-old WT and Lmna^–/– hearts. (B) PCA of proteins in 2-week-old WT and Lmna^–/– hearts. (C) Venn diagram depicting differential and overlapping differentially expressed proteins (DEPs) in 1-month-old and 2-week-old WT and Lmna^–/– hearts (upper); 257 DEPs in 1 month consistent with the trend of change at 2 weeks of age (lower). (D) Gene Set Enrichment Analysis showing enriched top 10 up-regulated and down-regulated Kyoto Encyclopedia of Genes and Genomes pathways. (E) Gene Set Enrichment Analysis showing enriched top 10 up-regulated and down-regulated Reactome pathways. (F) Heatmaps summarizing differentially expressed mitochondrial respiratory chain Ⅰ, Ⅲ, and Ⅳ assembly proteins. (G) Representative Western blot images of electron transport chain complex subunits (complexes I-V) in 1-month-old WT and Lmna^–/– hearts. (H) Quantification of electron transport chain complex I to V expressions normalized to β-actin (n = 3). Data are presented as mean ± SD and were compared by using Student’s t-test (panel H). ∗P < 0.05, ∗∗P < 0.01. ATP = adenosine triphosphate; cAMP = cyclic adenosine monophosphate; ns = no significant difference; PPAR = peroxisome proliferator–activated receptor; other abbreviations as in Figure 1.

Figure 3

Lamin A/C Deficiency Leads to Aberrant Mitochondrial Structure and Function (A) Representative transmission electron microscopy images of myocardial mitochondria from 1-month-old WT and Lmna^–/– mice (scale bars: 5 μm and 1 μm). (B) Quantification of mitochondrial number per square micrometers (WT, n = 5; Lmna^–/–, n = 4), mitochondrial area (WT, n = 424; Lmna^–/–, n = 437), and cristae number per mitochondrion (WT, n = 330; Lmna^–/–, n = 354). (C) Quantification of the percentage of healthy, mildly injured, and severely injured mitochondria according to cristae density (WT, n = 5; Lmna^–/–, n = 4). (D) Representative transmission electron microscopy images of myocardial mitochondria from 2-week-old WT and Lmna^–/– mice. Scale bar: 5 μm and 1 μm. (E) Quantification of mitochondrial number per square micrometers (n = 3), mitochondrial area (WT, n = 238; Lmna^–/–, n = 248), and cristae number per mitochondrion (WT, n = 238; Lmna^–/–, n = 248). (F) Quantification of the percentage of healthy, mildly injured, and severely injured mitochondria according to cristae density (n = 3). (G and H) ATP levels in myocardial tissue of 1-month-old (G) and 2-week-old (H) WT and Lmna^–/– mice (n = 4). (I) Representative image of oxygen consumption rate (OCR) in negative control (NC) and lamin A/C knockdown (LKD) neonatal rat ventricular myocytes (NRVMs). (J) Quantification of basal respiration, ATP production, maximal respiration, and spare respiratory capacity (n = 6). (K) Representative fluorescence images showing mitochondrial membrane potential of NC and LKD NRVMs stained by tetramethylrhodamine methyl ester (TMRM) and MitoTracker Green (scale bar: 100 μm). (L) Quantification of TMRM intensity (n = 8). Data are presented as mean ± SD. Data in B (mito number), C, E (mito number), F, G, J, and L were compared by using the Student’s t-test, and data in B (mito area, cristae number), E (mito area, cristae number), and H were compared by using the Mann-Whitney U test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations as in Figures 1 and 2.

Figure 4

Lamin A/C Deficiency Causes Impaired Mitochondrial Biogenesis, Imbalanced Fission and Fusion, and Defected Mitophagy (A and B) Heatmaps summarizing DEPs related to mitochondrial quality control (MQC) in 1-month-old (A) and 2-week-old (B) WT and Lmna^–/– hearts. (C to F) Representative Western blot images of MQC-related proteins in 1-month-old (C) and 2-week-old (E) WT and Lmna^–/– hearts. Quantification of MQC-related proteins expressions normalized to β-actin in 1-month-old (D) and 2-week-old (F) WT and Lmna^–/– hearts (n = 3). Data are presented as mean ± SD and were compared by using Student’s t-tests (D and F). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. DRP1 = dynamin-related protein 1; MFN2 = mitofusin 2; PGC1α, peroxisome proliferator–activated receptor gamma coactivator 1-alpha; PINK1 = PTEN-induced kinase 1; SIRT1 = sirtuin 1; other abbreviations as in Figures 1 and 2.

Figure 5

Overexpression of SIRT1 Prevents Cardiac Mitochondrial Injury Caused by Lamin A/C Deficiency (A) Representative image of mitochondrial OCR in NC and LKD NRVMs transfected with adenovirus expressing SIRT1-3×FLAG-eGFP (Ad-SIRT1) or adenovirus expressing enhanced green fluorescent protein (eGFP) only (Ad-Control). (B) Quantification of basal respiration, ATP production, maximal respiration, and spare respiratory capacity (n = 5). (C) Representative fluorescence images showing mitochondrial membrane potential of NC and LKD NRVMs transfected with Ad-SIRT1-3×FLAG-eGFP or Ad-eGFP stained by TMRM. Scale bar: 100 μm. (D) Quantification of TMRM intensity (n = 8). (E) Adeno-associated virus (AAV) vector was intraperitoneally injected in neonatal 1-3 days old WT and Lmna^–/– mice. Hearts were collected at 1 month for further analysis. (F) Representative transmission electron microscopy images of myocardial mitochondria from 1-month-old WT and Lmna^–/– mice pretreated with AAV-CMV-SIRT1-mNeonGreen (AAV-SIRT1) or AAV-CMV-mNeonGreen (AAV-Control). Scale bars: 5 μm and 1 μm. (G) Quantification of mitochondrial number per square micrometers (WT+AAV-Control, Lmna^–/–+AAV-Control, Lmna^–/–+AAV-SIRT1, n = 4; WT+AAV-SIRT1, n = 3), mitochondrial area (WT+AAV-Control, n = 318; WT+AAV-SIRT1, n = 263; Lmna^–/–+AAV-Control, n = 420; Lmna^–/–+AAV-SIRT1, n = 397), and cristae number per mitochondrion (WT+AAV-Control, n = 319; WT+AAV-SIRT1, n = 263; Lmna^–/–+AAV-Control, n = 420; Lmna^–/–+AAV-SIRT1, n = 397). (H) Quantification of the percentage of healthy, mildly injured, and severely injured mitochondria according to cristae density (WT+AAV-Control, Lmna^–/–+AAV-Control, Lmna^–/–+AAV-SIRT1, n = 4; WT+AAV-SIRT1, n = 3). Data are presented as mean ± SD. Data in B, D, G (mito number), and H were compared by using an analysis of variance with the Tukey post hoc test, and data in G (mito area, cristae number) were compared by using the Kruskal-Wallis test followed by the Dunn post hoc test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations as in Figure 1, Figure 2, Figure 3, Figure 4.

Figure 6

SIRT1-Based AAV Injection Alleviates Cardiac Dysfunction and Prolongs Survival of Lmna^–/– Mice (A) Representative echocardiography of WT and Lmna^–/– mice pretreated with AAV-SIRT1 or AAV-Control. (B) LW/BW of WT and Lmna^–/– mice pretreated with AAV-SIRT1 or AAV-Control (WT+AAV-Control, WT+AAV-SIRT1, n = 5; Lmna^–/–+AAV-Control, Lmna^–/–+AAV-SIRT1, n = 4). (C) Quantification of LVEF, FS, and HR (n = 6). (D) Quantification of LVESV/BW, LVEDV/BW, LVIDs/BW, and LVIDd/BW (n = 6). (E) Representative HE staining (scale bar: 1 mm) and Masson trichrome staining (scale bar: 100 μm) images of the ventricular transverse section of WT and Lmna^–/– mice pretreated with AAV-SIRT1 or AAV-Control. (F) Quantification of the percentage of collagen in myocardial tissue (n = 4). (G) Kaplan-Meier curves of Lmna^–/– mice pretreated with AAV-Control (median survival 36.5 days) or AAV-SIRT1 (median survival 48.5 days) (WT+AAV-Control, Lmna^–/–+AAV-Control, Lmna^–/–+AAV-SIRT1, n = 8; WT+AAV-SIRT1, n = 7; Breslow generalized Wilcoxon test). Data are presented as mean ± SD. Data in B, C (HR), D, and F were compared by using an analysis of variance with a Tukey post hoc test; data in C (LVEF and FS) were compared by using the Kruskal-Wallis test with the Dunn post hoc test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations as in Figures 1, 3, 4, and 5.

Figure 7

Lamin A/C Maintain Mitochondrial Bioenergetics Through SIRT1-PARKIN Axis (A) Representative Western blot images of MQC-related proteins and electron transport chain (ETC) complex subunits (complex I-V) in WT and Lmna^–/– mice pretreated with AAV-SIRT1 or AAV-Control. (B and C) Quantification of MQC-related proteins (B) and complex I to V (C) expressions normalized to β-actin (n = 3). (D) Representative image of OCR in LKD NRVMs transfected with Ad-Control or Ad-SIRT1, and lamin A/C and PARKIN knockdown (LPKD) NRVMs transfected with Ad-SIRT1. (E) Quantification of ATP production, maximal respiration, and spare respiratory capacity (n = 5). Data are presented as mean ± SD and were compared by using analysis of variance with a Tukey post hoc test (B, C, and E). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Abbreviations as in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5.