Left ventricular concentric hypertrophy with cardiac magnetic resonance imaging improves risk stratification in patients with Duchenne muscular dystrophy: a prospective cohort study.

BACKGROUND: The development of left ventricular (LV) remodeling has been associated with an increased cardiovascular risk and cardiogenic death, and different patterns of remodeling result in varying levels of prognosis. OBJECTIVE: To investigate the association between different patterns of LV remodeling and clinical outcomes in the preclinical stage of patients with Duchenne muscular dystrophy (DMD). MATERIALS AND METHODS: A total of 148 patients with DMD and 43 sex- and age-matched healthy participants were enrolled. We used the four-quadrant analysis method to investigate LV remodeling based on cardiac magnetic resonance (MR) imaging. Kaplan-Meier curves were generated to illustrate the event-free survival probability stratified by the LV remodeling pattern. Cox regression models were constructed and compared to evaluate the incremental predictive value of the LV remodeling pattern. RESULTS: During the median follow-up period of 2.2 years, all-cause death, cardiomyopathy, and ventricular arrhythmia occurred in 5, 35, and 7 patients, respectively. LV concentric hypertrophy (hazard ratio 2.91, 95% confidence interval 1.47-5.75, P=0.002) was an independent predictor of composite endpoint events. Compared to the model without LV concentric hypertrophy, the model with LV concentric hypertrophy had significant incremental predictive value (chi-square value 33.5 vs. 25.2, P=0.004). CONCLUSION: Age and late gadolinium enhancement positivity were positively correlated with clinical outcomes according to the prediction models. LV concentric hypertrophy was also an independent predictor for risk stratification and provided incremental value for predicting clinical outcomes in the preclinical stage of patients with DMD.

Clinical importance of changes in magnetic resonance biomarkers for Duchenne muscular dystrophy.

OBJECTIVE: Magnetic resonance (MR) measures of muscle quality are highly sensitive to disease progression and predictive of meaningful functional milestones in Duchenne muscular dystrophy (DMD). This investigation aimed to establish the reproducibility, responsiveness to disease progression, and minimum clinically important difference (MCID) for multiple MR biomarkers at different disease stages in DMD using a large natural history dataset. METHODS: Longitudinal MR imaging and spectroscopy outcomes and ambulatory function were measured in 180 individuals with DMD at three sites, including repeated measurements on two separate days (within 1 week) in 111 participants. These data were used to calculate day-to-day reproducibility, responsiveness (standardized response mean, SRM), minimum detectable change, and MCID. A survey of experts was also performed. RESULTS: MR spectroscopy fat fraction (FF), as well as MR imaging transverse relaxation time (MRI-T2 ), measures performed in multiple leg muscles, and had high reproducibility (Pearson's R > 0.95). Responsiveness to disease progression varied by disease stage across muscles. The average FF from upper and lower leg muscles was highly responsive (SRM > 0.9) in both ambulatory and nonambulatory individuals. MCID estimated from the distribution of scores, by anchoring to function, and via expert opinion was between 0.01 and 0.05 for FF and between 0.8 and 3.7 ms for MRI-T2 . INTERPRETATION: MR measures of FF and MRI T2 are reliable and highly responsive to disease progression. The MCID for MR measures is less than or equal to the typical annualized change. These results confirm the suitability of these measures for use in DMD and potentially other muscular dystrophies.

Hybrid QUS Radiomics: A Multimodal-Integrated Quantitative Ultrasound Radiomics for Assessing Ambulatory Function in Duchenne Muscular Dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a neuromuscular disorder that affects ambulatory function. Quantitative ultrasound (QUS) imaging, utilizing envelope statistics, has proven effective in diagnosing DMD. Radiomics enables the extraction of detailed features from QUS images. This study further proposes a hybrid QUS radiomics and explores its value in characterizing DMD. METHODS: Patients (n = 85) underwent ultrasound examinations of gastrocnemius through Nakagami, homodyned K (HK), and information entropy imaging. The hybrid QUS radiomics extracted, selected, and integrated the retained features derived from each QUS image for classification of ambulatory function using support vector machine. Nested five fold cross-validation of the data was conducted, with the rotational process repeated 50 times. The performance was assessed by averaging the areas under the receiver operating characteristic curve (AUROC). RESULTS: Radiomics enhanced the average AUROC of B-scan, Nakagami, HK, and entropy imaging to 0.790, 0.911, 0.869, and 0.890, respectively. By contrast, the hybrid QUS radiomics using HK and entropy images for diagnosing ambulatory function in DMD patients achieved a superior average AUROC of 0.971 (p < 0.001 compared with conventional radiomics analysis). CONCLUSIONS: The proposed hybrid QUS radiomics incorporates microstructure-related backscattering information from various envelope statistics models to effectively enhance the performance of DMD assessment.

Abbreviated cardiac magnetic resonance imaging versus echocardiography for interval assessment of systolic function in Duchenne muscular dystrophy: patient satisfaction, clinical utility, and image quality.

PURPOSE: Poor acoustic windows make interval assessment of systolic function in patients with (Duchenne Muscular Dystrophy) DMD by echocardiography (echo) difficult. Cardiac magnetic resonance imaging (CMR) can be challenging in DMD patients due to study duration and patient discomfort. We developed an abbreviated CMR (aCMR) protocol and hypothesized that aCMR would compare favorably to echo in image quality and clinical utility without significant differences in exam duration, patient satisfaction, and functional measurements. METHODS: DMD patients were recruited prospectively to undergo echo and aCMR. Modalities were compared with a global quality assessment score (GQAS), clinical utility score (CUS), and patient satisfaction score (PSS). Results were compared using Wilcoxon signed-rank tests, Spearman correlations, intraclass correlations, and Bland-Altman analyses. RESULTS: Nineteen DMD patients were included. PSS scores and exam duration were equivalent between modalities, while CUS and GQAS scores favored aCMR. ACMR scored markedly higher than echo in RV visualization and assessment of atrial size. Older age was negatively correlated with echo GQAS and CUS scores, as well as aCMR PSS scores. Higher BMI was positively correlated with aCMR GQAS scores. Nighttime PPV requirement and non-ambulatory status were correlated with worse echo CUS scores. Poor image quality precluding quantification existed in five (26%) echo and zero (0%) aCMR studies. There was moderate correlation between aCMR and echo for global circumferential strain and left ventricular four chamber global longitudinal strain. CONCLUSION: The aCMR protocol resulted in improved clinical relevance and quality scores relative to echo, without significant detriment to patient satisfaction or exam duration.

Reporting of paediatric osteoporotic vertebral fractures in Duchenne muscular dystrophy and potential impact on clinical management: the need for standardised and structured reporting.

BACKGROUND: In boys with Duchenne muscular dystrophy (DMD), initiation of bisphosphonate is recommended upon identification of moderate or severe vertebral fractures, even if asymptomatic. Clear radiological reporting is important for consistency of clinical interpretation and management. OBJECTIVES: To audit radiology reports of spine imaging for vertebral fracture assessment in DMD, and assess potential impact on diagnosis and management. MATERIALS AND METHODS: Lateral thoracolumbar spine imaging (71 lateral spine radiographs and 13 lateral dual energy absorptiometry spine image) in 84 boys with DMD performed across two centres. Anonymised radiology reports by paediatric radiologists were circulated to two neuromuscular clinicians and two endocrinologists. Clinicians determined if there was vertebral fracture, no vertebral fracture, or unclear interpretation. Endocrinologists also determined if bisphosphonate was indicated. A single observer (a clinician with expertise in vertebral fracture assessment) performed vertebral fracture assessment in 37 images and re-reported using a structured format. Structured reports were re-circulated to the four clinicians to re-evaluate the degree of concordance in clinical diagnosis of vertebral fracture and treatment decisions with bisphosphonate. RESULTS: The term "fracture" was used in 25/84 (30%) radiology reports and only in 8/43 (19%) with description of vertebral body abnormalities. Fracture grading was included in 7/43 (16%) radiology reports. Diagnostic concordance by the clinicians was noted in 36/84 (43%). Unclear interpretation was noted in 22% to 51% based on radiology reports. No unclear interpretation was noted with structured reports. Complete diagnostic (37/37, 100%) and treatment (37/37, 100%) concordance was noted with the structured reports, whereas complete diagnostic and treatment concordance was noted in only 16/37 (43%) and 17/37 (46%) of the radiology reports, respectively. CONCLUSION: Only a third of radiology reports of spine imaging in DMD explicitly used the terminology "fracture". Grading was only noted in a small percentage. Variability in diagnostic interpretation by clinicians may lead to differing management plans. As identification of vertebral fracture is a trigger for treatment, developing reporting guidelines for paediatric vertebral fracture assessment will improve care. A structured template should be introduced for radiological reporting of paediatric vertebral fracture assessment.

Left ventricular strain-volume loops and myocardial fibrosis in pediatric patients with Duchenne muscular dystrophy.

BACKGROUND: The left ventricular strain-volume loop (SVL) combines changes in global longitudinal strain (GLS) and LV volume across a cardiac cycle, providing insight into cardiac dynamics. This study explored the association between left ventricular SVL and presence of fibrosis, assessed with late gadolinium enhancement, in patients with Duchenne muscular dystrophy (DMD). METHODS AND RESULTS: 34 pediatric patients with DMD were included. Feature tracking analysis was used to assess endocardial GLS and volumetric measurements to construct the SVL. Mean age at the time of assessment was 14 ± 3 and 11 ± 2 years old (p < 0.01) in the group with (n = 18) versus without fibrosis (n = 16), respectively. Left ventricular ejection fraction was not significantly different between groups (fibrosis: 56.4 ± 3.8% versus without fibrosis: 54.0 ± 6.3%, p = 0.18). After adjusting for age, the late diastolic slope of the SVL was significantly associated with presence of fibrosis (OR 0.39 [95% CI 0.18-0.85]; area under the receiver operating characteristic curve: 0.83 [95% CI 0.70-0.97]) No significant association was observed for peak strain and fibrosis (OR 1.15 [95% CI 0.86-1.546]). CONCLUSION: A lower late diastolic slope of the left ventricular SVL, related to the interplay between longitudinal deformation and volume changes late in diastole, is associated with presence of myocardial fibrosis in pediatric patients with DMD.

A novel biomarker of fibrofatty replacement in dystrophinopathies identified by integrating transcriptome, magnetic resonance imaging, and pathology data.

BACKGROUND: We aimed to analyse genome-wide transcriptome differences between Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) patients and identify biomarkers that correlate well with muscle magnetic resonance imaging (MRI) and histological fibrofatty replacement in both patients, which have not been reported. METHODS: One hundred and one male patients with dystrophinopathies (55 DMD and 46 BMD) were enrolled. Muscle-derived genome-wide RNA-sequencing was performed in 31 DMD patients, 29 BMD patients, and 11 normal controls. Fibrofatty replacement was scored on muscle MRI and histological levels in all patients. A unique pipeline, single-sample gene set enrichment analysis combined with Spearman's rank correlations (ssGSEA-Cor) was developed to identify the most correlated gene signature for fibrofatty replacement. Quantitative real-time PCR (qRT-PCR) analysis, western blot analysis, and single-nucleus RNA-sequencing (snRNA-seq) were performed in the remaining patients to validate the most correlated gene signature. RESULTS: Comparative transcriptomic analysis revealed that 31 DMD muscles were characterized by a significant increase of inflammation/immune response and extracellular matrix remodelling compared with 29 BMD muscles (P < 0.05). The ssGSEA-Cor pipeline revealed that the gene set of CDKN2A and CDKN2B was the most correlated gene signature for fibrofatty replacement (histological rs  = 0.744, P < 0.001; MRI rs  = 0.718, P < 0.001). Muscle qRT-PCR confirmed that CDKN2A mRNA expression in both 15 DMD (median = 25.007, P < 0.001) and 12 BMD (median = 5.654, P < 0.001) patients were significantly higher than that in controls (median = 1.101), while no significant difference in CDKN2B mRNA expression was found among DMD, BMD, and control groups. In the 27 patients, muscle CDKN2A mRNA expression respectively correlated with muscle MRI (rs  = 0.883, P < 0.001) and histological fibrofatty replacement (rs  = 0.804, P < 0.001) and disease duration (rs  = 0.645, P < 0.001) and North Star Ambulatory Assessment total scores (rs  = -0.698, P < 0.001). Muscle western blot analysis confirmed that both four DMD (median = 2.958, P < 0.05) and four BMD (median = 1.959, P < 0.01) patients had a significantly higher level of CDKN2A protein expression than controls (median = 1.068). The snRNA-seq analysis of two DMD muscles revealed that CDKN2A was mainly expressed in fibro-adipogenic progenitors, satellite cells, and myoblasts. CONCLUSIONS: We identify CDKN2A expression as a novel biomarker of fibrofatty replacement, which might be a new target for antifibrotic therapy in dystrophinopathies.

Derivation and validation of diagnostic models for myocardial fibrosis in duchenne muscular dystrophy: assessed by multi-parameter cardiovascular magnetic resonance.

BACKGROUND: Gadolinium-enhanced cardiovascular magnetic resonance (CMR) is the most widely used approach for diagnosing myocardial fibrosis with late gadolinium enhancement (LGE) in cardiomyopathy associated with Duchenne muscular dystrophy. Given the limitations and safety of gadolinium use, we wanted to develop and evaluate multi-parametric pre-contrast CMR models for the diagnosis of LGE and investigate whether they could be utilised as surrogates for LGE in DMD patients. METHODS: A total of 136 DMD patients were prospectively recruited and separated into LGE - and LGE + groups. In the first subset of patients (derivation cohort), regression models for the diagnosis of LGE were built by logistic regression using pre-contrast sequence parameters. In a validation cohort of other patients, the models' performances were evaluated. RESULTS: EF, native T1 and longitudinal strain alone, as well as their combinations form seven models. The model that included EF, native T1 and longitudinal strain had the best diagnostic value, but there was no significant difference in diagnostic accuracy among the other models except EF. In the validation cohort, the diagnosis outcomes of models were moderate consistent with the existence of LGE. The longitudinal strain outperformed the other models in terms of diagnostic value (sensitivity: 83.33%, specificity: 54.55%). CONCLUSIONS: Pre-contrast sequences have a moderate predictive value for LGE. Thus, pre-contrast parameters may be considered only in a specific subset of DMD patients who cannot cooperate for long-time examinations and have contradiction of contrast agent to help predict the presence of LGE. TRIAL REGISTRATION NUMBER (TRN): ChiCTR1800018340 DATE OF REGISTRATION: 20180107.

Electrical impedance myography detects dystrophin-related muscle changes in mdx mice.

BACKGROUND: The lack of functional dystrophin protein in Duchenne muscular dystrophy (DMD) causes chronic skeletal muscle inflammation and degeneration. Therefore, the restoration of functional dystrophin levels is a fundamental approach for DMD therapy. Electrical impedance myography (EIM) is an emerging tool that provides noninvasive monitoring of muscle conditions and has been suggested as a treatment response biomarker in diverse indications. Although magnetic resonance imaging (MRI) of skeletal muscles has become a standard measurement in clinical trials for DMD, EIM offers distinct advantages, such as portability, user-friendliness, and reduced cost, allowing for remote monitoring of disease progression or response to therapy. To investigate the potential of EIM as a biomarker for DMD, we compared longitudinal EIM data with MRI/histopathological data from an X-linked muscular dystrophy (mdx) mouse model of DMD. In addition, we investigated whether EIM could detect dystrophin-related changes in muscles using antisense-mediated exon skipping in mdx mice. METHODS: The MRI data for muscle T2, the magnetic resonance spectroscopy (MRS) data for fat fraction, and three EIM parameters with histopathology were longitudinally obtained from the hindlimb muscles of wild-type (WT) and mdx mice. In the EIM study, a cell-penetrating peptide (Pip9b2) conjugated antisense phosphorodiamidate morpholino oligomer (PPMO), designed to induce exon-skipping and restore functional dystrophin production, was administered intravenously to mdx mice. RESULTS: MRI imaging in mdx mice showed higher T2 intensity at 6 weeks of age in hindlimb muscles compared to WT mice, which decreased at ≥ 9 weeks of age. In contrast, EIM reactance began to decline at 12 weeks of age, with peak reduction at 18 weeks of age in mdx mice. This decline was associated with myofiber atrophy and connective tissue infiltration in the skeletal muscles. Repeated dosing of PPMO (10 mg/kg, 4 times every 2 weeks) in mdx mice led to an increase in muscular dystrophin protein and reversed the decrease in EIM reactance. CONCLUSIONS: These findings suggest that muscle T2 MRI is sensitive to the early inflammatory response associated with dystrophin deficiency, whereas EIM provides a valuable biomarker for the noninvasive monitoring of subsequent changes in skeletal muscle composition. Furthermore, EIM reactance has the potential to monitor dystrophin-deficient muscle abnormalities and their recovery in response to antisense-mediated exon skipping.

Brain Abnormalities in Becker Muscular Dystrophy: Evaluation by Voxel-Based DTI and Morphometric Analysis.

BACKGROUND AND PURPOSE: Although various neuropsychological problems in Becker muscular dystrophy have attracted attention, there have been few related neuroimaging studies. We investigated brain abnormalities in patients with Becker muscular dystrophy using 3D T1WI and DTI. MATERIALS AND METHODS: MR images were obtained for 30 male patients and 30 age-matched healthy male controls. We classified patients into Dp140+ and Dp140- subgroups based on their predicted dystrophin Dp140 isoform expression and performed voxel-based comparisons of gray and white matter volumes and DTI metrics among the patients, patient subgroups, and controls. ROI-based DTI analyses were also performed. RESULTS: Significantly decreased fractional anisotropy was observed in the left planum temporale and right superior parietal lobule compared between the Becker muscular dystrophy and control groups. In the Dp140- subgroup, decreased fractional anisotropy was observed in the left planum temporale, but no significant changes were seen in the Dp140+ subgroup. The ROI-based analysis obtained the same results. No significant differences were evident in the gray or white matter volumes or the DTI metrics other than fractional anisotropy between the groups. CONCLUSIONS: A DTI metric analysis is useful to detect white-matter microstructural abnormalities in Becker muscular dystrophy that may be affected by the Dp140 isoform expression.

Acoustic radiation force impulse shear wave elastography quantifies upper limb muscle in patients with Duchenne muscular dystrophy.

We investigated whether the upper limb muscle stiffness quantified by the acoustic radiation force impulse shear wave elastography (ARFI/SWE) is a potential biomarker for age-related muscle alteration and functional decline in patients with Duchenne muscular dystrophy (DMD). 37 patients with DMD and 30 typically developing controls (TDC) were grouped by age (3-8, 9-11, and 12-18 years). ARFI/SWE measured the biceps and deltoid muscle's shear wave velocities (SWVs). Performance of Upper Limb Module (PUL 1.2 module) assessed muscle function in DMD patients. Mann Whitney test compared muscle SWVs between DMD and TDC, stratified by three age groups. We used analysis of variance with Bonferroni correction to compare muscle SWVs between DMD and TDC and correlated muscle SWVs with PUL results in the DMD group. Results showed that the SWVs of biceps differentiated DMD patients from TDC across age groups. Younger DMD patients (3-8 years) exhibited higher SWVs (p = 0.013), but older DMD patients (12-18 years) showed lower SWVS (p = 0.028) than same-aged TDC. DMD patients had decreasing biceps SWVs with age (p < 0.001), with no such age effect in TDC. The SWVs of deltoid and biceps positively correlated with PUL scores (r = 0.527 âˆ¼ 0.897, P < 0.05) and negatively correlated with PUL timed measures (r = -0.425 âˆ¼ -0.542, P < 0.05) in DMD patients. Our findings suggest that ARFI/SWE quantifying the SWVs in upper limb muscle could be a potential biomarker to differentiate DMD from TDC across ages and that DMD patients showed age-related muscle alteration and limb functional decline.

Longitudinal changes in magnetic resonance imaging biomarkers of the gluteal muscle groups and functional ability in Duchenne muscular dystrophy: a 12-month cohort study.

BACKGROUND: Quantitative magnetic resonance imaging (MRI) is considered an objective biomarker of Duchenne muscular dystrophy (DMD), but the longitudinal progression of MRI biomarkers in gluteal muscle groups and their predictive value for future motor function have not been described. OBJECTIVE: To explore MRI biomarkers of the gluteal muscle groups as predictors of motor function decline in DMD by characterizing the progression over 12 months. MATERIALS AND METHODS: A total of 112 participants with DMD were enrolled and underwent MRI examination of the gluteal muscles to determine fat fraction and longitudinal relaxation time (T1). Investigations were based on gluteal muscle groups including flexors, extensors, adductors, and abductors. The North Star Ambulatory Assessment and timed functional tests were performed. All participants returned for follow-up at an average of 12 months and were divided into two subgroups (functional stability/decline groups) based on changes in timed functional tests. Univariable and multivariable logistic regression methods were used to explore the risk factors associated with future motor function decline. RESULTS: For the functional decline group, all T1 values decreased, while fat fraction values increased significantly over 12 months (P<0.05). For the functional stability group, only the fat fraction of the flexors and abductors increased significantly over 12 months (P<0.05). The baseline T1 value was positively correlated with North Star Ambulatory Assessment and negatively correlated with timed functional tests at the 12-month follow-up (P<0.001), while the baseline fat fraction value was negatively correlated with North Star Ambulatory Assessment and positively correlated with timed functional tests at the 12-month follow-up (P<0.001). Multivariate regression showed that increased fat fraction of the abductors was associated with future motor function decline (model 1: odds ratio [OR]=1.104, 95% confidence interval [CI]: 1.026~1.187, P=0.008; model 2: OR=1.085, 95% CI: 1.013~1.161, P=0.019), with an area under the curve of 0.874. CONCLUSION: Fat fraction of the abductors is a powerful predictor of future motor functional decline in DMD patients at 12 months, underscoring the importance of focusing early on this parameter in patients with DMD.

Comparison of two corticosteroid regimens on brain volumetrics in patients with Duchenne muscular dystrophy.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is a neuromuscular disorder in which many patients also have neurobehavioral problems. Corticosteroids, the primary pharmacological treatment for DMD, have been shown to affect brain morphology in other conditions, but data in DMD are lacking. This study aimed to investigate the impact of two corticosteroid regimens on brain volumetrics in DMD using magnetic resonance imaging (MRI). METHODS: In a cross-sectional, two-center study, T1-weighted MRI scans were obtained from three age-matched groups (9-18 years): DMD patients treated daily with deflazacort (DMDd, n = 20, scan site: Leuven), DMD patients treated intermittently with prednisone (DMDi, n = 20, scan site: Leiden), and healthy controls (n = 40, both scan sites). FSL was used to perform voxel-based morphometry analyses and to calculate intracranial, total brain, gray matter, white matter, and cerebrospinal fluid volumes. A MANCOVA was employed to compare global volumetrics between groups, with site as covariate. RESULTS: Both patient groups displayed regional differences in gray matter volumes compared to the control group. The DMDd group showed a wider extent of brain regions affected and a greater difference overall. This was substantiated by the global volume quantification: the DMDd group, but not the DMDi group, showed significant differences in gray matter, white matter, and cerebrospinal fluid volumes compared to the control group, after correction for intracranial volume. INTERPRETATION: Volumetric differences in the brain are considered part of the DMD phenotype. This study suggests an additional impact of corticosteroid treatment showing a contrast between pronounced alterations seen in patients receiving daily corticosteroid treatment and more subtle differences in those treated intermittently.

Left ventricular deformation and myocardial fibrosis in pediatric patients with Duchenne muscular dystrophy.

BACKGROUND: Left ventricular (LV) strain and rotation are emerging functional markers for early detection of LV dysfunction and have been associated with the burden of myocardial fibrosis in several disease states. This study examined the association between LV deformation (i.e., LV strain and rotation) and extent and location of LV myocardial fibrosis in pediatric patients with Duchenne muscular dystrophy (DMD). METHODS AND RESULTS: 34 pediatric patients with DMD underwent cardiovascular magnetic resonance (CMR) with late gadolinium enhancement (LGE) to assess LV myocardial fibrosis. Offline CMR feature-tracking analysis was used to assess global and segmental longitudinal and circumferential LV strain, and LV rotation. Patients with fibrosis (n = 18, 52.9%) were older than those without fibrosis (14 ± 3 years (yrs) vs 11 ± 2 yrs., p = 0.01). There was no significant difference in LV ejection fraction (LVEF) between subjects with and without fibrosis (54 ± 6% vs 56 ± 4%, p = 0.18). However, lower endocardial global circumferential strain (GCS), but not LV rotation, was associated with presence of fibrosis (adjusted Odds Ratio 1.25 [95% CI 1.01-1.56], p = 0.04). Both GCS and global longitudinal strain correlated with the extent of fibrosis (r = .52, p = 0.03 and r = .75, p < 0.01, respectively). Importantly, segmental strain did not seem to correspond to location of fibrosis. CONCLUSION: A lower global, but not segmental, strain is associated with presence and extent of LV myocardial fibrosis in pediatric DMD patients. Therefore, strain parameters might detect structural myocardial alterations, however currently more research is needed to evaluate its value (e.g., prognostic) in clinical practice.

In vivo shear wave elasticity imaging for assessment of diaphragm function in muscular dystrophy.

Duchenne muscular dystrophy (DMD) causes patients to suffer from ambulatory disability and cardiorespiratory failure, the latter of which leads to premature death. Due to its role in respiration, the diaphragm is an important muscle for study. A common method for evaluating diaphragm function is ex vivo force testing, which only allows for an end point measurement. In contrast, ultrasound shear wave elastography imaging (US-SWEI) can assess diaphragm function over time; however, US-SWEI studies in dystrophic patients to date have focused on the limbs without preclinical studies. In this work, we used US-SWEI to estimate the shear wave speed (SWS) in diaphragm muscles of healthy (WT) mice, mdx mice, and mdx mice haploinsufficient for utrophin (mdx-utr) at 6 and 12 months of age. Diaphragms were then subjected to ex vivo force testing and histological analysis at 12 months of age. Between 6 and 12 months, a 23.8% increase in SWS was observed in WT mice and a 27.8% increase in mdx mice, although no significant difference was found in mdx-utr mice. Specific force generated by mdx-utr diaphragms was lower than that of WT diaphragms following twitch stimulus. A strong correlation between SWS and collagen deposition was observed, as well as between SWS and muscle fiber size. Together, these data demonstrate the ability of US-SWEI to evaluate dystrophic diaphragm functionality over time and predict the biochemical and morphological make-up of the diaphragm. Additionally, our results highlight the advantage of US-SWEI over ex vivo testing by obtaining longitudinal measurements in the same subject. STATEMENT OF SIGNIFICANCE: In DMD patients, muscles experience cycles of regeneration and degeneration that contribute to chronic inflammation and muscle weakness. This pathology only worsens with time and leads to muscle wasting, including in respiratory and cardiac muscles. Because respiratory failure is a major contributor to premature death in DMD patients, the diaphragm muscle is an important muscle to evaluate and treat over time. Currently, diaphragm function is assessed using ex vivo force testing, a technique that only allows measurement at sacrifice. In contrast, ultrasonography, particularly shear wave elasticity imaging (USSWEI), is a promising tool for longitudinal assessment; however, most US-SWEI in DMD patients aimed for limb muscles only with the absence of preclinical studies. This work broadens the applications of US-SWE imaging by demonstrating its ability to track properties and function of dystrophic diaphragm muscles longitudinally in multiple dystrophic mouse models.

A Protocol for Simultaneous In Vivo Imaging of Cardiac and Neuroinflammation in Dystrophin-Deficient MDX Mice Using [18F]FEPPA PET.

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder caused by dystrophin loss-notably within muscles and the central neurons system. DMD presents as cognitive weakness, progressive skeletal and cardiac muscle degeneration until pre-mature death from cardiac or respiratory failure. Innovative therapies have improved life expectancy; however, this is accompanied by increased late-onset heart failure and emergent cognitive degeneration. Thus, better assessment of dystrophic heart and brain pathophysiology is needed. Chronic inflammation is strongly associated with skeletal and cardiac muscle degeneration; however, neuroinflammation's role is largely unknown in DMD despite being prevalent in other neurodegenerative diseases. Here, we present an inflammatory marker translocator protein (TSPO) positron emission tomography (PET) protocol for in vivo concomitant assessment of immune cell response in hearts and brains of a dystrophin-deficient mouse model [mdx:utrn(+/-)]. Preliminary analysis of whole-body PET imaging using the TSPO radiotracer, [18F]FEPPA in four mdx:utrn(+/-) and six wildtype mice are presented with ex vivo TSPO-immunofluorescence tissue staining. The mdx:utrn(+/-) mice showed significant elevations in heart and brain [18F]FEPPA activity, which correlated with increased ex vivo fluorescence intensity, highlighting the potential of TSPO-PET to simultaneously assess presence of cardiac and neuroinflammation in dystrophic heart and brain, as well as in several organs within a DMD model.

Innovative Computerized Dystrophin Quantification Method Based on Spectral Confocal Microscopy.

Several clinical trials are working on drug development for Duchenne and Becker muscular dystrophy (DMD and BMD) treatment, and, since the expected increase in dystrophin is relatively subtle, high-sensitivity quantification methods are necessary. There is also a need to quantify dystrophin to reach a definitive diagnosis in individuals with mild BMD, and in female carriers. We developed a method for the quantification of dystrophin in DMD and BMD patients using spectral confocal microscopy. It offers the possibility to capture the whole emission spectrum for any antibody, ensuring the selection of the emission peak and allowing the detection of fluorescent emissions of very low intensities. Fluorescence was evaluated first on manually selected regions of interest (ROIs), proving the usefulness of the methodology. Later, ROI selection was automated to make it operator-independent. The proposed methodology correctly classified patients according to their diagnosis, detected even minimal traces of dystrophin, and the results obtained automatically were statistically comparable to the manual ones. Thus, spectral imaging could be implemented to measure dystrophin expression and it could pave the way for detailed analysis of how its expression relates to the clinical course. Studies could be further expanded to better understand the expression of dystrophin-associated protein complexes (DAPCs).

Brain magnetic resonance imaging in the DE50-MD dog model of Duchenne muscular dystrophy reveals regional reductions in cerebral gray matter.

BACKGROUND: Duchenne muscular dystrophy is a X-linked disease characterized by severe and progressive muscle weakness, alongside cognitive impairment and a range of neurobehavioral disorders secondary to brain dystrophin deficiency. Duchenne muscular dystrophy patients have reduced cerebral gray matter and altered white matter ultrastructure (detected by magnetic resonance imaging) compared to age-matched controls. METHODS: We studied the DE50-MD canine model of Duchenne muscular dystrophy, which is deficient in full length brain dystrophin (Dp427) isoforms and has a neurocognitive phenotype. Eight DE50-MD and 6 age-matched littermate wild type male dogs underwent serial brain magnetic resonance imaging from 14 to 33 months of age. RESULTS: Reduced regional gray matter was detected in DE50-MD dogs compared with wildtype, including the piriform lobe, hippocampus and cingulate gyrus. Lateral ventricle volume was larger in DE50-MD dogs. Differences did not progress over time. White matter volume did not differ between DE50-MD and wildtype dogs. There was no difference in brain nor cranial vault volume between DE50-MD and wildtype dogs. CONCLUSION: Dystrophin deficiency in the canine brain results in structural changes that likely contribute to the neurocognitive phenotype.