Dmpk gene deletion or antisense knockdown does not compromise cardiac or skeletal muscle function in mice

Abstract

Myotonic dystrophy type 1 (DM1) is a genetic disorder in which dominant-active DM protein kinase (DMPK) transcripts accumulate in nuclear foci, leading to abnormal regulation of RNA processing. A leading approach to treat DM1 uses DMPK-targeting antisense oligonucleotides (ASOs) to reduce levels of toxic RNA. However, basal levels of DMPK protein are reduced by half in DM1 patients. This raises concern that intolerance for further DMPK loss may limit ASO therapy, especially since mice with Dmpk gene deletion reportedly show cardiac defects and skeletal myopathy. We re-examined cardiac and muscle function in mice with Dmpk gene deletion, and studied post-maturity knockdown using Dmpk-targeting ASOs in mice with heterozygous deletion. Contrary to previous reports, we found no effect of Dmpk gene deletion on cardiac or muscle function, when studied on two genetic backgrounds. In heterozygous knockouts, the administration of ASOs reduced Dmpk expression in cardiac and skeletal muscle by > 90%, yet survival, electrocardiogram intervals, cardiac ejection fraction and muscle strength remained normal. The imposition of cardiac stress by pressure overload, or muscle stress by myotonia, did not unmask a requirement for DMPK. Our results support the feasibility and safety of using ASOs for post-transcriptional silencing of DMPK in muscle and heart.

Figure 1.

DMPK knockdown in heart. (A) Heterozygous Dmpk knockout mice (+/-) on the FVB background received subcutaneous injection of saline or ASO 486178 (ASO) at 50mg/kg/wk (mpk/wk) for 1 month. Immunoblots for DMPK protein were performed on apical heart tissue and quantified, compared to wild-type (WT) and homozygous knockout (-/-) littermates (n =  3–4 per group). GAPDH was used as loading control. (B, C) Quantification of DMPK protein in (B) apical heart tissue of C57Bl6 +/- mice given saline or ASO for 1 month at 50mpk/wk (n =  3 per group); or (C) isolated cardiac myocytes of FVB +/- mice given saline or ASO at 50mpk/wk for 1 month then 50mpk biweekly for 2 months, compared to WT controls (n =  4–5 per group). (D) As in A, except that saline or ASO was started at 2 months of age and continued up to age 11–12 months, compared to age-matched WT and -/- littermates (n =  5–6 per group). (E) As in D, except on C57Bl6 background, (n =  4–5 per group). * = ANOVA P <  0.05 and T-test p ≤ 0.0084. † = T-test P <  0.05.

Figure 2.

No alterations of ECG intervals with Dmpk loss. (A–C) Shown are surface ECG measurements of (A) heart rate; (B) PR interval; and (C) QRS duration in 6- and 10-month old FVB +/- mice given saline or ASO, with WT and -/- littermate controls (n =  9–11 per group; 6 males and 3–5 females per group). (D–F) Similar to A–C, except on C57Bl6 background (n = 11-12 per group; 5-6 males and 6 females per group). (G) Representative plot of heart rate from an implantable telemeter recording. Grey bars denote 15-min high- and low-activity segments chosen for signal averaging and conduction interval analysis. (H) Representative ECG traces from surface (top) and implantable telemetry (bottom). Scale bars are 120 msec. (I-K) Measurements from implantable telemetry of (I) heart rate; (J) PR interval; and (K) QRS duration in 11–12-month-old FVB Dmpk mice (n =  4–6 per group). ANOVA P-value is shown above each data set.

Figure 3.

Dmpk silencing does not induce systolic dysfunction or cardiac fibrosis. (A) Ejection fraction determined by echocardiography in 6- and 10-month-old FVB Dmpk mice (n =  9–11 per group; 6 males and 3–5 females per group) and in C57Bl6 Dmpk mice (n =  11–12 per group; 5–6 males and 6 females per group). (B) Heart weight normalized to tibia length (HW/TL) in 11–12-month-old FVB Dmpk mice (n =  5-6 per group) and in 10–11-month-old C57Bl6 Dmpk mice (n =  4–5 per group). ANOVA P-Value above each data set. (C-F) Representative images of heart cryosections stained with picrosirius red, from 18-month old FVB WT, saline-injected Dmpk^+/-, ASO-injected Dmpk^+/-, and Dmpk ^-/- littermates, respectively. Low picrosirius staining in all sections indicates a lack of pathological fibrosis. Scale bar is 50 µm.

Figure 4.

Effect of Dmpk silencing on cardiac response to pressure overload stress. FVB +/- (Het) and -/- (KO) mice were given saline or ASO at 50 mpk/wk starting at 2 months of age. Baseline measurements of (A) ejection fraction and (B) estimated LV mass were taken at age 3 months, along with age-matched WT and uninjected KO controls. Transverse aortic constriction (TAC) banding was performed after baseline measurements. Ejection fraction and LV mass were monitored at 3, 5–6, and 8 weeks following TAC banding (n =  17–19 for WT, Het+Saline, and Het+ASO at baseline to 5-6 weeks. n =  8–11 for KO, KO+Saline, and KO+ASO and for all groups at 8 weeks.) ANOVA P-value as shown above each data set. * = t-test P <  0.003, ** = t-test P <  0.00005. (C) Lung weight normalized to body weight (LW/BW mg/g) in FVB Dmpk mice 9 weeks post-TAC, with age-matched non-TAC controls. Dotted line denotes threshold for decompensated heart failure (LW/BW > 8mg/g). Fisher’s Exact Test showed no significant increase of lung weight in any group compared to WT controls.

Figure 5.

Dmpk silencing does not affect force generation or induce dystrophic changes in limb muscle. (A) Four-limb grip strength measurements of peak force from 8–9-month-old FVB Dmpk mice (n =  8–12), and 8–9-month-old C57Bl6 Dmpk mice (n =  11–12). ASO injections were initiated at age 2 months. (B) Ex vivo measurements of isometric, specific tetanic force and (C) force-frequency response of extensor digitorum longus (EDL) muscles from 10 to 11-month-old C57Bl6 Dmpk mice (n =  3–4 mice, or 5–8 muscles). ANOVA P-value above each data set. (D-G) Representative images of H&E from quadriceps muscle in 18-month old FVB WT, saline-injected Dmpk^+/-, ASO-injected Dmpk^+/-, and Dmpk ^-/- littermates, respectively. Scale bar is 25 µm.