Obscurin deficiency leads to compensated dilated cardiomyopathy and increased arrhythmias

Abstract

Obscurin is a large muscle protein whose multiple functions include providing mechanical strength to the M-band and linking the sarcomere to the sarcoplasmic reticulum. Mutations in obscurin are linked to various forms of muscle diseases. This study compares cardiac function in a murine model of obscurin deletion (KO) with wild-type (WT) in vivo and ex vivo. Echocardiography showed that KO hearts had larger (+20%) end-diastolic and end-systolic volumes, reduced fractional shortening, and impaired ejection fraction, consistent with dilated cardiomyopathy. However, stroke volume and cardiac output were preserved due to increased end-diastolic volume. Morphological analyses revealed reduced sarcoplasmic reticulum volume, with preserved T-tubule network. While myofilament function was preserved in isolated myofibrils and skinned trabeculae, experiments in intact trabeculae revealed that Obscn KO hearts compared with WT displayed (1) reduced active tension at high frequencies and during resting-state contractions, (2) impaired positive inotropic and lusitropic response to β-adrenergic stimulation (isoproterenol 0.1 μM), and (3) faster mechanical restitution, suggesting reduced sarcoplasmic reticulum refractoriness. Intracellular [Ca2+]i measurements showed reduced peak systolic and increased diastolic levels in KO versus WT cardiomyocytes. Western blot experiments revealed lower SERCA and phospholamban (PLB) expression and reduced PLB phosphorylation in KO mice. While action potential parameters and conduction velocity were unchanged, β-adrenergic stimulation induced more frequent spontaneous Ca2+ waves and increased arrhythmia susceptibility in KO compared with WT. Taken together, these findings suggest that obscurin deletion, in adult mice, is linked to compensated dilated cardiomyopathy, altered E-C coupling, impaired response to inotropic agents, and increased propensity to arrhythmias.

Figure 1.

Echocardiographic measurements. (A) Representative parasternal long-axis and short-axis views of the LV at end-diastole from 8-mo-old WT and Obscn KO male mice. Horizontal scale bars equal 1 mm. (B) Thickness of the IVS measured at end-diastole (left) and at end-systole (right) in WT and Obscn KO mice. (C) LV volumes calculated using the Simpson technique at end-diastole (left) and end-systole (right) in mice from the two study groups. (D) Systolic thickening of the IVS (left) and LV ejection fraction (LV-EF%, right), expressed as the percentage of the diastolic values, measured in mice from the two cohorts. (E) SV calculated from the Simpson volumes and CO. (F) Ratio between E and A waves (E/A ratio, left) and isoVolumic relaxation time (IVRT, right) from transmitral blood flow velocity curves recorded using pulsed-wave Doppler echocardiography in apical views. Statistical tests: One-way ANOVA with Tukey’s correction. Data are means ± SEM from 7 Obscn KO and 10 WT mice, respectively. *0.05 > P > 0.01; **0.01 > P > 0.001. IVS, interventricular septum; SV, stroke volume; CO, cardiac output.

Figure 2.

Histological analyses. (A) Representative hematoxylin and eosin staining of cross sections from 8-mo-old WT and Obscn KO hearts (n = 3). Bar = 1 mm. (B) Thickness (μm ± SD) of left and right walls, and of the interventricular septum of WT and Obscn KO hearts. Heart weight, expressed as the percentage of body weight, is shown in the bottom right panel. (C) Representative image of immunofluorescence staining for the detection of collagen III (scale bar equals 1 mm). (D) Representative confocal images from isolated LV cardiomyocytes stained with Di-3-ANEPPDHQ. The horizontal bar equals 20 μm. (E) TT density, as calculated using the Ttorg ImageJ plug-in, in cardiomyocytes from the two groups. (F) Representative electron micrographs of WT and Obscn KO hearts. +, *, and # indicate fully, partial, and not extended l-SR, respectively. Scale bar = 1 µm. (G) Analysis of l-SR extension in WT and Obscn KO myofibrils. Quantification of fully (full), partial (part), and not extended l-SR in WT and Obscn KO hearts is reported as the percentage for each type of l-SR. ***P < 0.001 versus WT, as determined by Student’s t test. (H) Representative electron micrographs of WT and Obscn KO TT and analysis of TT area in WT and Obscn KO hearts. ****P < 0.0001 versus WT, as determined by a two-tailed Mann–Whitney test. Scale bar = 250 µm. l-SR, longitudinal SR.

Figure 3.

Steady-state isometric twitches and short-term interval–force relationship. (A) Representative force recordings from trabeculae of 8-mo-old WT and Obscn KO male mice, stimulated at 5 Hz, in the absence and in the presence of Iso 0.1 µM. (B) Relationship between active twitch force and stimulation frequency (0.1–8 Hz) in trabeculae from WT and Obscn KO mice, in the absence and in the presence of Iso 0.1 µM. (C and D) (C) Time to peak and (D) time from peak to 50% relaxation measured in steady-state twitches at different stimulation frequencies (0.1–8 Hz) in trabeculae from WT and Obscn KO mice, in the absence and in the presence of Iso 0.1 µM. Data are means ± SEM from N = 5 WT mice, n = 10 trabeculae; and N = 5 Obscn KO mice, n = 12 trabeculae. Statistical tests: one-way ANOVA with Tukey’s correction with *0.05 > P > 0.01; **0.01 > P > 0.001.

Figure 4.

Ca ²⁺ recirculation fraction and SERCA/PLB expression. (A) Representative Ca²⁺ recirculation fraction estimated by the decline of potentiated beats following a period of high stimulation rate in trabeculae from trabeculae of 8-mo-old WT and Obscn KO male mice, stimulated at 5 Hz, in the absence and in the presence of Iso 0.1 µm. (B) Representative western blots for total SERCA, PLB, phospho-PLB at serine 16, and GAPDH. (C) Protein expression levels of WT (n = 5) and Obscn KO (n = 5) hearts excised from 8-mo-old male mice. The intensity of individual bands was quantified following normalization to that of GAPDH. The mean relative intensity of WT samples was set at 1. **P < 0.01 and ***P < 0.001 versus WT, as determined by Student’s t test. Source data are available for this figure: SourceData F4.

Figure 5.

Mechanical restitution and SR-Ca ²⁺ leakage. (A) Mechanical restitution protocol: representative traces from WT and Obscn KO trabeculae prepared from 8-mo-old male mice: steady-state stimulation, 1 Hz; premature interval, 250–600 ms. (B) Restitution curves show the fractional recovery of force (percentage of 1-Hz steady-state peak force) in response to the premature stimulus plotted against the premature interval. (C) Post-postextrasystolic potentiation is calculated by normalization of the postextrasystolic beat and the last regular beat following the extrasystole. (D) Representative traces showing the stimulation pause protocol used to elicit spontaneous Ca²⁺ events in WT and Obscn KO cardiomyocytes at basal conditions and in the presence of Iso 0.1 µmol/liter (Iso). Notably, Obscn KO cardiomyocytes showed frequent Ca²⁺ oscillations (red arrows). (E) Frequency of spontaneous Ca²⁺ waves and spontaneous Ca²⁺ transients during stimulation pauses in WT and Obscn KO cardiomyocytes at basal conditions and in the presence of 0.1 µmol/liter Iso. Data are the mean ± SEM from n = 55 WT (N = 5 mice), n = 35 Obscn KO (N = 5 mice) cardiomyocytes. Statistical tests: one-way ANOVA with Tukey’s correction with *0.05 > P > 0.01.

Figure 6.

Intracellular Ca ²⁺ measurements in intact ventricular cardiomyocytes. (A) Representative superimposed Ca²⁺ transients elicited at 5 Hz in WT and Obscn KO cardiomyocytes. (B) Mean ± SE of diastolic (left) and systolic (right) Ca²⁺ levels, expressed as arbitrary units of fluorescence intensity, during steady-state stimulation at different frequencies in n = 55 WT cardiomyocytes (N = 5 mice), n = 35 Obscn KO cardiomyocytes (N = 5 mice). *P < 0.05.

Figure 7.

AP kinetics, conduction, and arrhythmogenicity in the Langendorff-perfused Obscn KO mouse heart. (A) Representative fluorescence images showing WT and Obscn KO mouse heart. Scale bar = 2 mm. The hearts were electrically paced at the apex (yellow bolt). (B) Fluorescent signals (∆F/F) were extracted from the LV (black ROI: CTRL, blue ROI: Obscn KO) during a burst of stimuli at 5 Hz. (C) Average AP upstroke kinetics (TTP) and AP duration at 50% and 90% of repolarization (APD50 and APD90, respectively) in the LV free wall of WT (black) and Obscn KO (blue) hearts. (D) Average CV (m/s) in CTRL and Obscn KO LV free walls. (E) VT inducibility in WT and Obscn KO hearts. Data were collected from seven WT to eight Obscn KO mouse hearts and reported as the mean ± SEM. Student’s t test analysis was performed with *0.05 > P > 0.01 and **0.01 > P > 0.001. CV, conduction velocity; TTP, time to peak; VT, ventricular tachycardia.