Figure 1.
Deletion of both Nesprin 1 and 2 causes decreased cardiac wall thickness and impaired cardiac function.
(A–D) Left ventricular anterior wall during diastole and systole (LVAWd and LVAWs), Left ventricular posterior wall during diastole and systole (LVPWd and LVPWs) at 5, 10, 25 and 52 weeks. (E) % Fractional Shortening (%FS) at 5, 10, 25, 52 weeks. (F) Heart Rate (HR) at 5, 10, 25, 52 weeks. N = 6–10 mice per condition/timepoint. ANOVA with a post hoc Bonferroni test *p<0.05, N/C = No significant change vs. Wild Type.
Figure 2.
Ablation both Nesprin 1 and 2 alters cardiac morphology, reactivates fetal gene program and activates profibrotic genes.
(A) and (B), 10 week old mice hearts: Morphological analyses of heart weight (HW)/body weight (BW) or HW/tibia length(TL). N = 10–11samples per condition (C–F), Real Time PCR analyses of mRNA expression levels for (C) ANP (D) ßMHC (E) αMHC (F) Procol1α1 (G) Procol3α1 N = 3–4 per condition. N/C = No significant change, ANOVA with a post hoc Bonferroni test #p<0.01.
Figure 3.
Histological and TUNEL analyses of Nesprin 1 and/or 2 ablation.
(A) Representative 100× H&E images of indicated hearts. Black arrows = Myocyte nuclei. Black bar = 80 µm. (B) and (C), Representative 40× images of (B), Picrosirius Red (Brown = nuclei, red = collagen) or (C) Masson Trichrome (Red = Myocardium, Blue = Collagen, Black = Nuclei). Black bar = 200 µm. (D) TUNEL analyses of Nesprin 1 and/or 2 knockout hearts at 5 Weeks or 10 Weeks. N = 3 per condition. N/C = No significant change, ANOVA with a post hoc Bonferroni test #p<0.01.
Figure 4.
Cardiomyocyte nuclear morphology is altered in response to loss of Nesprin 1 and/or 2.
(A) Representative 40× images of isolated cardiomyocytes (Blue = DAPI, Green = Phalloidin). White bar = 15 µm. (B–G) Morphological analyses of cardiomyocytes examining (B) Myocyte area (C) Nuclear circularity (D) Nuclear area (E) Nuclear perimeter (F) Nuclear length or (G) Nuclear distance. N = 200–300 cells per condition, from 3–4 mice. ANOVA with a post hoc Bonferroni test *p<0.05, #p<0.01.
Figure 5.
Examination of cardiomyocyte nuclear ultrastructure in response to loss of Nesprin 1 and/or 2.
(A–D) Representative images of postnatal day 1 myocyte nuclei from (A) Wild Type (B) nesprin 1f/f;nesprin 2+/+;Nkx2.5Cre (C),nesprin 1f/f;nesprin 2−/− or (D), nesprin 1f/f;nesprin 2−/−;Nkx2.5Cre hearts. (E–H) Representative images of 10 week old myocyte nuclei from (E) Wild Type (F) nesprin 1f/f;nesprin 2+/+;Nkx2.5Cre (G) nesprin 1f/f;nesprin 2−/− or (H) nesprin 1f/f;nesprin 2−/−;Nkx2.5Cre hearts. Black Bars = 1 µm. Red Arrows highlight electron dense heterochromatin defects in 10 week old nesprin 1f/f;nesprin 2−/−;Nkx2.5Cre hearts.
Figure 6.
Real Time PCR analyses of LINC complex and LINC complex associated factors.
(A–E) Real Time PCR analyses of (A) Lamin A/C (B) Lamin B1 (C)Lamin B2 (D)Emerin (E)SUN1 (F)SUN2. Real Time PCR analyses are representative of N = 4–5 cardiomyocyte isolations. ANOVA with a post hoc Bonferroni test. N/C = No significant change.
Figure 7.
Examination of biomechanical gene response in isolated neonatal cardiomyocytes from Wild Type, nesprin 1f/f;nesprin 2+/+; Nkx2.5Cre, nesprin 1f/f;nesprin 2−/− or nesprin 1f/f;nesprin 2−/−;Nkx2.5Cre hearts.
(A–E)Real Time PCR analyses of mRNA expression levels for (A) egr-1 (B) c-jun (C) iex-1 (D) c-fos and (E) c-myc. N = 3–5 stretchers/isolations per condition. ANOVA with a post hoc Bonferroni test N/C = No significant change, *p<0.05, #p<0.01.
Figure 8.
Model of Nesprin 1 and/or 2 loss in cardiomyocytes.
(A and B) Diagrammatic representation of (A) Wild Type and (B) Nesprin 1 and 2 double knockout cardiomyocyte nuclei. (A) Wild Type nuclei are anchored to the sarcomeric structure via Nesprin 1 and 2. Application of 14% anisotropic loading results in normal cellular biomechanical gene response (egr-1, c-jun, iex-1, c-fos and c-myc.). Cardiac development and nuclear positioning/morphology are also normal in these cells. (B) Loss of both Nesprin 1 and 2 causes ablation of biomechanical gene response upon application of strain. Hearts from these mice develop cardiomyopathy with fibrosis, and have altered nuclear positioning and morphology.