Cardiovascular phenotype of the Dmdmdx rat – a suitable animal model for Duchenne muscular dystrophy

Fig. 1.. Cardiac function and morphology assessment by transthoracic echocardiography. (A-E) Ejection fraction (EF; A), left ventricular (LV) end-systolic diameter (LVESD; B), LV end-diastolic diameter (LVEDD; C), ratio between early mitral inflow velocity and mitral annular early diastolic velocity (E/E′; D) and mean pulmonary artery pressure (mPAP; E) in wt and Dmdmdx rats at 3, 7 and 9 months of age. Data are expressed as mean±s.d.; n=8 wt and n=7 Dmdmdx. *P<0.05, **P<0.01 and ***P<0.001, using unpaired Student's t-test at defined age (3, 7 and 9 months).

Fig. 2.. RT-qPCR data on LV tissue from wt and Dmdmdx rats at 9 months of age. NOX4, NADPH oxidase 4; MRTFA, myocardin-related transcription factor A; MRTFB, myocardin-related transcription factor B; SERCA2a, sarcoplasmic reticulum Ca2+-ATPase 2a; SLC, sarcolipin; PLB, phospholamban; nNOS, neuronal nitric oxide synthase; ACE1, angiotensin-converting enzyme; AT1R, angiotensin II type 1 receptor. Data are expressed as mean±s.d.; n=10 wt and n=8 Dmdmdx. *P<0.05, **P<0.01, ***P<0.001, using unpaired Student's t-test.

Fig. 3.. Histopathological evaluation of cardiac fibrosis and inflammation from wt and Dmdmdx rats at 9 months of age. (A) Representative Masson-Goldner staining of LV sections from wt and Dmdmdx rats at 9 months of age. Blue indicates fibrosis. (B) Quantitative data showing the extent of LV and right ventricular fibrosis. (C) Collagen I and III mRNA expression in myocardium. (D,E) High-magnification photomicrographs show increased levels of CD68+ macrophages (D) and tenascin-C (E) expression in Dmdmdx rat hearts compared to wt rat hearts. Scale bars: 1 mm (overview) and 50 μm (enlarged sections). Data are expressed as mean±s.d.; n=7 wt and n=7 Dmdmdx in histology; n=11 wt and n=10 Dmdmdx in RT-qPCR. *P<0.05, ***P<0.001, using unpaired Student's t-test.

Fig. 4.. Vascular reactivity and inflammation in lung endothelial cells of wt and Dmdmdx rats at 9 months of age. (A) Endothelium-dependent relaxation in response to acetylcholine (ACh) in aortic rings from wt and Dmdmdx rats. (B) Relaxations induced by sodium nitroprusside (SNP) in aortic rings from wt and Dmdmdx rats. Data are expressed as mean±s.d.; n=7 wt and n=9 Dmdmdx. *P<0.05, using two-way ANOVA. (C) mRNA expression of Sod1, Nox4, Vcam1, ACE1, Il1b, Mrtfa and Mrtfb in wt and dystrophic lung endothelial cells. SOD1, superoxide dismutase; NOX4, NADPH oxidase 4; VCAM1, vascular cell adhesion molecule 1; ACE1, angiotensin-converting enzyme; IL-1β, interleukin 1 beta; MRTFA, myocardin-related transcription factor A; MRTFB, myocardin-related transcription factor B. n=4 wt and n=5 Dmdmdx animal. *P<0.05, **P<0.01, using unpaired Student's t-test.

Fig. 5.. Angiotensin-converting enzyme (ACE) activity in wt and Dmdmdx rats. (A-C) ACE activity in kidney (A), lung (B) and LV (C) tissue samples from wt and Dmdmdx rats at 9 months of age. Data are expressed as mean±s.d.; n=9-10 wt and n=8 Dmdmdx. *P<0.05, using unpaired Student's t-test.

Fig. 6.. Electrically stimulated and caffeine-induced intracellular Ca2+ transients in wt and Dmdmdx rat ventricular cardiomyocytes. (A,B) Representative time courses of Fluo-4 fluorescence reporting rises in cytosolic Ca2+ concentration during electrical field stimulation, at 0.2 Hz frequency, in a single wt (A) and Dmdmdx (B) myocyte. (C) Comparison of mean Ca2+ peak fluorescence relative to baseline (F/F0) between wt and Dmdmdx myocytes. Each data point represents a single cell, and values are expressed as means±s.d. [n=67 and n=53 for wt (four animals) and Dmdmdx (four animals) myocytes, respectively]. (D) Comparison of the Ca2+ transient decay kinetics in wt and Dmdmdx cardiomyocytes. The decay of the electrically induced Ca2+ signal following the rapid initial rise was fitted with a single exponential function to derive τ-values. (E,F) Time courses of Fluo-4 fluorescence reporting rises in cytosolic Ca2+ concentration during application of 20 mM caffeine in a wt (E) and Dmdmdx (F) myocyte. The gray bars indicate the time period of superfusion with bath solution containing caffeine. (G) Comparison of mean Ca2+ peak fluorescence, elicited by caffeine application, relative to baseline…

Fig. 7.. Regulation of electrically induced (0.2 Hz) Ca2+ transients by beta-adrenergic stimulation and inhibition of nNOS signaling in wt and Dmdmdx ventricular cardiomyocytes. (A) Comparison of the effect of superfusion with bath solution containing 100 nM isoprenaline on mean Ca2+ peak fluorescence relative to baseline (F/F0) in wt and Dmdmdx myocytes [n=63 and n=46 for wt (four animals) and Dmdmdx (four animals) myocytes, respectively]. (B) Comparison of the effect of superfusion with bath solution containing 100 µM of the nNOS inhibitor L-VNIO on mean Ca2+ peak fluorescence in wt and Dmdmdx myocytes [n=58 and n=31 for wt (three animals) and Dmdmdx (three animals) myocytes, respectively].

Fig. 8.. L-type Ca2+ channel expression and function in wt and Dmdmdx ventricular cardiomyocytes. (A) Cav1.2 Ca2+ channel expression and localization in single isolated wt and Dmdmdx ventricular cardiomyocytes. Two typical examples of Cav1.2 immunostaining in wt and Dmdmdx myocytes are shown. (B-D) L-type Ca2+ channel properties in wt and Dmdmdx ventricular cardiomyocytes. (B) Typical examples of original traces of barium currents through Ca2+ channels in a wt and Dmdmdx myocyte elicited by the pulse protocol displayed on top. (C) The inward current peaks at the respective applied membrane potentials, derived from a series of such experiments, were divided by the cell capacitance to obtain the current density-voltage relationship. Values are expressed as means±s.d. [n=10 and n=13 for wt (four animals) and Dmdmdx (four animals) myocytes, respectively]. (D) Barium current decay kinetics (expressed as τ-values derived from single exponential fits of the current decay after channel activation) at different membrane potentials were compared between wt and Dmdmdx myocytes. *P=0.046, using unpaired Student's t-test.