Figure 1.
Mice with moderate or severe cardiac dysfunction following four weeks of pressure overload.
Quantification of (A) fractional shortening, (B) ejection fraction and (C) left ventricular posterior wall thickness (LVPW) at four weeks post-TAC in control (CON), TAC moderate (TAC mod) and TAC severe (TAC sev) mice. Scatter plots demonstrate no overlap of fractional shortening and ejection fraction between TAC moderate and TAC severe groups. (D) Representative M-mode echocardiograms. N = 4–9 per group. *P<0.05 vs. control at four weeks post-TAC. †P<0.05. 1 way ANVOA followed by Fisher’s Post Hoc Test.
Table 1.
Echocardiography data of control and TAC mice at baseline and four weeks post-TAC.
Figure 2.
LNA-antimiR-34a is associated with modest improvements in cardiac remodeling in a moderate model of pressure overload.
(A) Representative heart images of control, TAC moderate LNA control (CON) and TAC moderate LNA-antimiR-34a (anti-34a) at dissection. Scale bar = 0.5 cm. (B-D) Graphs of HW/TL, AW/TL and LW/TL. N = 4–8 per group. ***P<0.001 vs. control, **P<0.01 vs. control, *P<0.05 vs. control, †P<0.05. 1 way ANVOA followed by Fisher’s Post Hoc Test.
Table 2.
Morphological data for control and TAC moderate and severe mice following four weeks of pressure overload and eight weeks after treatment with LNA-control or LNA-antimiR-34a.
Figure 3.
LNA-antimiR-34a maintains cardiac function in a moderate model of pressure overload, but not in a severe model.
(A) Graphs of HW/TL, AW/TL and LW/TL in control, TAC moderate (TAC mod) and TAC severe (TAC sev) LNA-control (c) and LNA-antimiR-34a (a) groups. N = 3–8 per group. ***P<0.001 vs. control, **P<0.01 vs. control, *P<0.05 vs. control, †P<0.05, §P<0.05 vs. TAC-moderate of same treatment group. 1 way ANOVA followed by Fisher’s Post Hoc Test (comparing all five groups). When performing 1 way ANOVA followed by Fisher’s Post Hoc Test on control and TAC moderate groups only (i.e. comparing three groups), n = 4–8 per group, ∧P<0.05 vs. control, ‡P<0.05. (B) Representative LV cross-sections stained with wheat germ agglutinin from control and TAC moderate and severe LNA-control and LNA-antimiR-34a mice, and quantification of cell area. Scale bar = 50 µM. Data are expressed as mean ± SEM. N = 3–5 per group. **P<0.01 vs. control, ***P<0.001 vs. control, §P<0.05. 1 way ANOVA followed by Fisher’s Post Hoc Test (comparing all five groups). (C) Quantification of fractional shortening at baseline (pre-surgery), four weeks post TAC (before LNA oligonucleotide administration) and 12 weeks post (i.e. eight weeks post treatment) and representative M-mode echocardiograms. N = 4–5 per group. *P<0.05 vs. baseline of the same groups and control at the same time point, †P<0.05, ‡P<0.05 vs. same group at 4 weeks post TAC, §P<0.05 vs. TAC-moderate at same time point. 1 way ANOVA followed by Fisher’s Post Hoc Test.
Table 3.
Echocardiography data of control and TAC mice at baseline, four weeks post-TAC and eight weeks after treatment with either LNA-control or LNA-antimiR-34a.
Figure 4.
Cardiac stress gene expression and fibrosis in moderate and severe models of pressure overload.
(A) Northern blots and quantification of Anp, Bnp, and Serca2a relative to Gapdh, in hearts of control (con), TAC moderate (mod), TAC severe (sev) mice dosed with either LNA-control (c) or LNA-antimiR-34a (a). For Northern blot of Anp, a light exposure (light exp) and dark exposure (dark exp) has been included. N = 3–5 per group. ***P<0.001 vs. control, **P<0.01 vs. control, *P<0.05 vs. control, §P<0.05, †P<0.05, 1 way ANOVA followed by Fisher’s Post Hoc Test (comparing all five groups). When performing 1 way ANOVA followed by Fisher’s Post Hoc Test on control and TAC severe groups only (comparing three groups), N = 3 per group, or control and TAC moderate groups only (comparing three groups), N = 3–5 per group, ∧P<0.05 vs. control. (B) qPCR of β-MHC relative to Hprt1 in control, TAC moderate and TAC severe mice dosed with LNA-control or LNA-antimiR-34a. N = 3–5 per group. *P<0.05 vs. control, §P<0.05, ‡P<0.05 vs. control, 1 way ANOVA followed by Fisher’s Post Hoc Test (comparing all 5 groups). (C) LV cross-sections stained with Masson’s trichrome and quantification of LV fibrosis in control, TAC moderate and TAC severe mice dosed with LNA-control or LNA-antimiR-34a. Scale = 200 µM. N = 4–5 per group. ***P<0.001 vs. control, §P<0.05 vs. TAC-moderate of the same treatment group, 1 way ANOVA followed by Fisher’s Post Hoc Test (comparing all five groups). When comparing control and TAC moderate groups only (comparing three groups), N = 4–5 per group, ∧P<0.05 vs. control, 1 way ANOVA followed by Fisher’s Post Hoc Test.
Figure 5.
Analysis of miR-34a target gene or protein expression.
(A) Representative Western blots and quantification of VEGF-A and VCL relative to GAPDH in hearts of control (con), TAC moderate (mod), TAC severe (sev) mice dosed with either LNA-control (c) or LNA-antimiR-34a (a). N = 3–5 per group. ∧P<0.05 vs. control when using 1 way ANOVA followed by Fisher’s Post Hoc test on severe group only. (B) qPCR analysis of Sirt1, Sema4b, Vegfb, Pofut1, PNUTS (Pppr10), Notch 1 and Cyclin D1 (Ccnd1) relative to Hprt1. N = 3–5 per group. ∧P<0.05 vs. control when using 1 way ANOVA followed by Fisher’s Post Hoc test on control and TAC severe groups only (comparing three groups).
Figure 6.
Expression of miR-34a, miR-34b and miR-34c in control and TAC mice.
qPCR of miR-34a, miR-b and miR-34c in hearts of control, TAC moderate and TAC severe mice. N = 3–5 per group. *P<0.05 vs. control, †P<0.05. 1 way ANOVA followed by Fisher’s Post Hoc test.