Regeneration of Nonhuman Primate Hearts With Human Induced Pluripotent Stem Cell-Derived Cardiac Spheroids.

BACKGROUND: The clinical application of human induced pluripotent stem cell-derived cardiomyocytes (CMs) for cardiac repair commenced with the epicardial delivery of engineered cardiac tissue; however, the feasibility of the direct delivery of human induced pluripotent stem cell-derived CMs into the cardiac muscle layer, which has reportedly induced electrical integration, is unclear because of concerns about poor engraftment of CMs and posttransplant arrhythmias. Thus, in this study, we prepared purified human induced pluripotent stem cell-derived cardiac spheroids (hiPSC-CSs) and investigated whether their direct injection could regenerate infarcted nonhuman primate hearts. METHODS: We performed 2 separate experiments to explore the appropriate number of human induced pluripotent stem cell-derived CMs. In the first experiment, 10 cynomolgus monkeys were subjected to myocardial infarction 2 weeks before transplantation and were designated as recipients of hiPSC-CSs containing 2×107 CMs or the vehicle. The animals were euthanized 12 weeks after transplantation for histological analysis, and cardiac function and arrhythmia were monitored during the observational period. In the second study, we repeated the equivalent transplantation study using more CMs (6×107 CMs). RESULTS: Recipients of hiPSC-CSs containing 2×107 CMs showed limited CM grafts and transient increases in fractional shortening compared with those of the vehicle (fractional shortening at 4 weeks after transplantation: 26.2±2.1%; 19.3±1.8%; P<0.05), with a low incidence of posttransplant arrhythmia. Transplantation of increased dose of CMs resulted in significantly greater engraftment and long-term contractile benefits (fractional shortening at 12 weeks after transplantation: 22.5±1.0%; 16.6±1.1%; P<0.01, left ventricular ejection fraction at 12 weeks after transplantation: 49.0±1.4%; 36.3±2.9%; P<0.01). The incidence of posttransplant arrhythmia slightly increased in recipients of hiPSC-CSs containing 6×107 CMs. CONCLUSIONS: We demonstrated that direct injection of hiPSC-CSs restores the contractile functions of injured primate hearts with an acceptable risk of posttransplant arrhythmia. Although the mechanism for the functional benefits is not fully elucidated, these findings provide a strong rationale for conducting clinical trials using the equivalent CM products.

Efficacy of exon-skipping therapy for DMD cardiomyopathy with mutations in actin binding domain 1.

Exon-skipping therapy is a promising treatment strategy for Duchenne muscular dystrophy (DMD), which is caused by loss-of-function mutations in the DMD gene encoding dystrophin, leading to progressive cardiomyopathy. In-frame deletion of exons 3-9 (Δ3-9), manifesting a very mild clinical phenotype, is a potential targeted reading frame for exon-skipping by targeting actin-binding domain 1 (ABD1); however, the efficacy of this approach for DMD cardiomyopathy remains uncertain. In this study, we compared three isogenic human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing Δ3-9, frameshifting Δ3-7, or intact DMD. RNA sequencing revealed a resemblance in the expression patterns of mechano-transduction-related genes between Δ3-9 and wild-type samples. Furthermore, we observed similar electrophysiological properties between Δ3-9 and wild-type hiPSC-CMs; Δ3-7 hiPSC-CMs showed electrophysiological alterations with accelerated CaMKII activation. Consistently, Δ3-9 hiPSC-CMs expressed substantial internally truncated dystrophin protein, resulting in maintaining F-actin binding and desmin retention. Antisense oligonucleotides targeting exon 8 efficiently induced skipping exons 8-9 to restore functional dystrophin and electrophysiological parameters in Δ3-7 hiPSC-CMs, bringing the cell characteristics closer to those of Δ3-9 hiPSC-CMs. Collectively, exon-skipping targeting ABD1 to convert the reading frame to Δ3-9 may become a promising therapy for DMD cardiomyopathy.

Mature human induced pluripotent stem cell-derived cardiomyocytes promote angiogenesis through alpha-B crystallin.

BACKGROUND: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can be used to treat heart diseases; however, the optimal maturity of hiPSC-CMs for effective regenerative medicine remains unclear. We aimed to investigate the benefits of long-term cultured mature hiPSC-CMs in injured rat hearts. METHODS: Cardiomyocytes were differentiated from hiPSCs via monolayer culturing, and the cells were harvested on day 28 or 56 (D28-CMs or D56-CMs, respectively) after differentiation. We transplanted D28-CMs or D56-CMs into the hearts of rat myocardial infarction models and examined cell retention and engraftment via in vivo bioluminescence imaging and histological analysis. We performed transcriptomic sequencing analysis to elucidate the genetic profiles before and after hiPSC-CM transplantation. RESULTS: Upregulated expression of mature sarcomere genes in vitro was observed in D56-CMs compared with D28-CMs. In vivo bioluminescence imaging studies revealed increased bioluminescence intensity of D56-CMs at 8 and 12 weeks post-transplantation. Histological and immunohistochemical analyses showed that D56-CMs promoted engraftment and maturation in the graft area at 12 weeks post-transplantation. Notably, D56-CMs consistently promoted microvessel formation in the graft area from 1 to 12 weeks post-transplantation. Transcriptomic sequencing analysis revealed that compared with the engrafted D28-CMs, the engrafted D56-CMs enriched genes related to blood vessel regulation at 12 weeks post-transplantation. As shown by transcriptomic and western blot analyses, the expression of a small heat shock protein, alpha-B crystallin (CRYAB), was significantly upregulated in D56-CMs compared with D28-CMs. Endothelial cell migration was inhibited by small interfering RNA-mediated knockdown of CRYAB when co-cultured with D56-CMs in vitro. Furthermore, CRYAB overexpression enhanced angiogenesis in the D28-CM grafts at 4 weeks post-transplantation. CONCLUSIONS: Long-term cultured mature hiPSC-CMs promoted engraftment, maturation and angiogenesis post-transplantation in infarcted rat hearts. CRYAB, which was highly expressed in D56-CMs, was identified as an angiogenic factor from mature hiPSC-CMs. This study revealed the benefits of long-term culture, which may enhance the therapeutic potential of hiPSC-CMs.

Intracoronary transplantation of pluripotent stem cell-derived cardiomyocytes: Inefficient procedure for cardiac regeneration.

Advances in stem cell biology have facilitated cardiac regeneration, and many animal studies and several initial clinical trials have been conducted using human pluripotent stem cell-derived cardiomyocytes (PSC-CMs). Most preclinical and clinical studies have typically transplanted PSC-CMs via the following two distinct approaches: direct intramyocardial injection or epicardial delivery of engineered heart tissue. Both approaches present common disadvantages, including a mandatory thoracotomy and poor engraftment. Furthermore, a standard transplantation approach has yet to be established. In this study, we tested the feasibility of performing intracoronary administration of PSC-CMs based on a commonly used method of transplanting somatic stem cells. Six male cynomolgus monkeys underwent intracoronary administration of dispersed human PSC-CMs or PSC-CM aggregates, which are called cardiac spheroids, with multiple cell dosages. The recipient animals were sacrificed at 4 weeks post-transplantation for histological analysis. Intracoronary administration of dispersed human PSC-CMs in the cynomolgus monkeys did not lead to coronary embolism or graft survival. Although the transplanted cardiac spheroids became partially engrafted, they also induced scar formation due to cardiac ischemic injury. Cardiac engraftment and scar formation were reasonably consistent with the spheroid size or cell dosage. These findings indicate that intracoronary transplantation of PSC-CMs is an inefficient therapeutic approach.

Restoring Dystrophin Expression with Duchenne Muscular Dystrophy Exon 45 Skipping in Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Induced pluripotent stem cell (iPSC)-based disease model is a useful tool that can represent the pathophysiology of patient organs that are inaccessible due to invasiveness. Here, we present a method to induce differentiation of Duchenne muscular dystrophy (DMD) patient-derived iPSCs into cardiomyocytes and restore dystrophin expression by exon skipping using antisense nucleic acids. This involves a 20-day multi-step culture process for differentiation to cardiomyocytes, followed by exon-skipping experiments. Additionally, RT-PCR, western blotting, and immunocytochemistry are used to confirm the restoration of dystrophin expression.

Increased predominance of the matured ventricular subtype in embryonic stem cell-derived cardiomyocytes in vivo.

Accumulating evidence suggests that human pluripotent stem cell-derived cardiomyocytes can affect "heart regeneration", replacing injured cardiac scar tissue with concomitant electrical integration. However, electrically coupled graft cardiomyocytes were found to innately induce transient post-transplant ventricular tachycardia in recent large animal model transplantation studies. We hypothesised that these phenomena were derived from alterations in the grafted cardiomyocyte characteristics. In vitro experiments showed that human embryonic stem cell-derived cardiomyocytes (hESC-CMs) contain nodal-like cardiomyocytes that spontaneously contract faster than working-type cardiomyocytes. When transplanted into athymic rat hearts, proliferative capacity was lower for nodal-like than working-type cardiomyocytes with grafted cardiomyocytes eventually comprising only relatively matured ventricular cardiomyocytes. RNA-sequencing of engrafted hESC-CMs confirmed the increased expression of matured ventricular cardiomyocyte-related genes, and simultaneous decreased expression of nodal cardiomyocyte-related genes. Temporal engraftment of electrical excitable nodal-like cardiomyocytes may thus explain the transient incidence of post-transplant ventricular tachycardia, although further large animal model studies will be required to control post-transplant arrhythmia.

High-amplitude fast activity in EEG: An early diagnostic marker in children with beta-propeller protein-associated neurodegeneration (BPAN).

OBJECTIVE: The early diagnosis of beta-propeller protein-associated neurodegeneration (BPAN) before distinct brain magnetic resonance imaging (MRI) findings of iron deposition occur remains challenging. This study examined whether children with BPAN have characteristic high-amplitude (>50 µV) fast activity (HAFA) on electroencephalography (EEG). METHODS: We conducted a retrospective analysis of EEG performed during childhood in five patients with BPAN. We also examined 143 EEGs from 59 patients with different etiologies, including epilepsy (n = 33), acute encephalopathy (n = 6), neurodevelopmental disorders (n = 5), non-epileptic events (n = 4), and others (n = 11). Trained electroencephalographers reviewed all of the EEGs. When excessive fast activity was observed, the amplitude, frequency, and locality were assessed. RESULTS: All five patients with BPAN underwent initial EEGs at 12-21 months old, and diffuse continuous HAFA (range 20-50 Hz) was observed on both awake and sleep EEGs. In the awake records, there was no clear posterior dominant rhythm in 4 of the 5 patients. Although 28% of the 143 EEGs had continuous excessive fast activity, mainly in the sleep records, only two (1.4%) exhibited HAFA when asleep, and their awake EEGs had clear posterior dominant rhythm. CONCLUSIONS: The EEGs of children with BPAN showed diffuse HAFA continuously when both awake and asleep, which is uncommon in children with other etiologies. SIGNIFICANCE: This study provides an important clue for the early diagnosis of BPAN.

Amelioration of intracellular Ca2+ regulation by exon-45 skipping in Duchenne muscular dystrophy-induced pluripotent stem cell-derived cardiomyocytes.

Duchenne muscular dystrophy (DMD) is a devastating muscle disorder caused by frameshift mutations in the DMD gene. DMD involves cardiac muscle, and the presence of ventricular arrhythmias or congestive failure is critical for prognosis. Several novel therapeutic approaches are being evaluated in ongoing clinical trials. Among them, exon-skipping therapy to correct frameshift mutations with antisense oligonucleotides is promising; however, their therapeutic efficacies on cardiac muscle in vivo remain unknown. In this study, we established induced-pluripotent stem cells (iPSCs) from T cells from a DMD patient carrying a DMD-exon 46-55 deletion, differentiated the iPSCs into cardiomyocytes, and treated them with phosphorodiamidate morpholino oligomers. The efficiency of exon-45 skipping increased in a dose-dependent manner and enabled restoration of the DMD gene product, dystrophin. Further, Ca2+-imaging analysis showed a decreased number of arrhythmic cells and improved transient Ca2+ signaling after exon skipping. Thus, exon-45 skipping may be effective for cardiac involvement in DMD patients harboring the DMD-exon 46-55 deletion.

Importance of long-term motor function evaluation after prednisolone treatment for Duchenne muscular dystrophy.

[Purpose] Motor function evaluation by physical therapists is considered a valuable tool to assess the progression of muscular dystrophies. Few reports have described long-term motor function assessment during the administration of corticosteroids such as prednisolone (PSL) in these patients. This study examined the importance of long-term non-invasive motor function evaluation in a series of 3 cases. [Participants and Methods] Three boys with Duchenne muscular dystrophy who were administered an identical PSL dosage regimen were retrospectively evaluated, and motor function tests were compared in them before and after an increase in PSL dosage. Regular feedback was obtained from the patients' mothers regarding their impressions of their child's motor function after the introduction of PSL. [Results] Motor function was conserved or significantly improved after an increase in dosage in all cases. Interestingly, subjective assessment by mothers revealed a perceived improvement only in case 1 without any changes reported in cases 2 or 3. [Conclusion] PSL was demonstrably effective for 2.5-5 years after initiating PSL treatment, although parental impressions varied. Thus, long-term non-invasive evaluation by physical therapists may provide important objective data regarding medication efficacy and disease progression. Future studies should include long-term testing results as an essential component of the discontinuation criteria for PSL.

The effect of wearing night splints for one year on the standing motor function of patients with Duchenne muscular dystrophy.

[Purpose] To investigate the effect of night splints on the standing motor function and ankle dorsiflexion angles of patients with Duchenne muscular dystrophy (DMD). [Subjects and Methods] Nine boys (age <11 years) with DMD were divided into the sufficiently-wearing group and the insufficiently-wearing group, according to how often they wore their splint for one year. We evaluated the changes between the pre-implementation and the one-year-after assessments of both the sufficiently-wearing group and the insufficiently-wearing group for the ankle dorsiflexion angle, North Star Ambulatory Assessment, 10-m running time, and time to stand from the floor. [Results] Only the left dorsiflexion angle of the ankle showed significantly difference for the sufficiently-wearing group. For other indicators, there were tendency toward improvement and maintenance in the sufficiently-wearing group. [Conclusion] The standing motor function improved significantly in some patients in the sufficiently-wearing group, suggesting that wearing night splints may promote the improvement and/or maintain of standing motor function in patients with DMD.

A Pediatric Case of Relapsing-Remitting Multiple Sclerosis Onset following Varicella Zoster Ophthalmicus with Optic Neuritis.

Some epidemiological studies have implied a pathogenetic association between varicella zoster virus (VZV) and multiple sclerosis (MS); this, however, remains controversial. The present report describes a case involving an immunocompetent 10-year-old girl who developed relapsing-remitting MS following the prolonged reactivation of VZV inside the first branch of the trigeminal nerve, exhibiting herpes zoster ophthalmicus with severe optic neuritis. Symptoms related to herpes zoster ophthalmicus and MS appeared consecutively in the 10-week period after the appearance of vesicles. This suggests that the onset of MS was triggered by some mechanism involving VZV reactivation in the first branch of the trigeminal nerve. To the best of our knowledge, this report is the first to describe a relationship between the onset of MS and herpes zoster ophthalmicus. Early diagnosis and aggressive antiviral therapy are important in cases of herpes zoster ophthalmicus to prevent the possible development of MS as well as visual impairment as sequela.

Clinical features of a female with WDR45 mutation complicated by infantile spasms: a case report and literature review.

We present a 3-year-old girl with beta-propeller protein-associated neurodegeneration (BPAN) who had a de novo heterozygous splice-site mutation of c.831-1G>C in WDR45 and developed infantile spasms; her onset age of infantile spasms was relatively late. Her infantile spasms and hypsarrhythmia disappeared promptly by adrenocorticotropic hormone therapy (CORTROSYN®Z, 0.0125mg/kg/day daily for 2weeks intramuscularly), though the administration of pyridoxal phosphate and valproic acid had poor efficacy. BPAN is known to be associated with various types of seizures, but there are few reports on infantile spasms, especially in females. To date, only 5 patients with BPAN have been reported to develop infantile spasms, and our patient is the second case in females. In this report, we showed that female patients with BPAN had milder phenotypic features than males: males developed intractable infantile spasms in early infancy, while females had treatable infantile spasms in late infancy.

Comparison of the phenotypes of patients harboring in-frame deletions starting at exon 45 in the Duchenne muscular dystrophy gene indicates potential for the development of exon skipping therapy.

Exon skipping therapy has recently received attention for its ability to convert the phenotype of lethal Duchenne muscular dystrophy (DMD) to a more benign form, Becker muscular dystrophy (BMD), by correcting the open reading frame. This therapy has mainly focused on a hot-spot (exons 45-55) mutation in the DMD gene. Exon skipping of an entire stretch of exons 45-55 is an approach applicable to 46.9% of DMD patients. However, the resulting phenotype is not yet fully understood. Here we examined the clinical profiles of 24 patients with BMD resulting from deletions starting at exon 45. The Δ45-55 group ranged in age from 2 to 87 years; no mortality was observed, and one patient was ambulatory at 79 years of age. The age at which patients became wheelchair-bound in the Δ45-48 group (18-88 years old) was approximately 50 years. Cardiomyopathy was well controlled by pharmaceuticals in both deletion groups. In contrast, the Δ45-47 and Δ45-49 groups exhibited more severe phenotypes than those with other mutations: the age at which patients in the Δ45-49 group became wheelchair-bound was around 30-40 years. Our study shows that clinical severity differs between each hot-spot deletion.

Usefulness of continuous actigraph monitoring in the assessment of the effect of corticosteroid treatment for Duchenne muscular dystrophy: a case report.

[Purpose] This study evaluated the effect of corticosteroid treatment on the daily activity of a patient with Duchenne muscular dystrophy using an actigraph and examined whether this method produces the same results as the conventional motor-function evaluation methods. [Subject and Methods] A patient with 5 year-old Duchenne muscular dystrophy was recruited. An actigraph was attached to his waist to measure the energy expenditure and the number of steps taken by him during a period of two weeks, 14 days before and 14 days after corticosteroid administration. The outcomes of these measurements were compared with the results of conventional motor-function evaluation methods-the 10-m run test, 6-minute walk test, and North Star Ambulatory Assessment-on his next visit. [Results] The actigraph data for energy expenditure and the number of steps taken correlated well with the results of the above-mentioned conventional motor-function tests, and the value of each data point improved after corticosteroid administration. [Conclusion] An actigraph was effectively used to non-invasively measure consecutive daily activity for four weeks. It was easily done and the results were consistent with conventional motor-function evaluation methods.

Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts.

Induced pluripotent stem cells (iPSCs) constitute a potential source of autologous patient-specific cardiomyocytes for cardiac repair, providing a major benefit over other sources of cells in terms of immune rejection. However, autologous transplantation has substantial challenges related to manufacturing and regulation. Although major histocompatibility complex (MHC)-matched allogeneic transplantation is a promising alternative strategy, few immunological studies have been carried out with iPSCs. Here we describe an allogeneic transplantation model established using the cynomolgus monkey (Macaca fascicularis), the MHC structure of which is identical to that of humans. Fibroblast-derived iPSCs were generated from a MHC haplotype (HT4) homozygous animal and subsequently differentiated into cardiomyocytes (iPSC-CMs). Five HT4 heterozygous monkeys were subjected to myocardial infarction followed by direct intra-myocardial injection of iPSC-CMs. The grafted cardiomyocytes survived for 12 weeks with no evidence of immune rejection in monkeys treated with clinically relevant doses of methylprednisolone and tacrolimus, and showed electrical coupling with host cardiomyocytes as assessed by use of the fluorescent calcium indicator G-CaMP7.09. Additionally, transplantation of the iPSC-CMs improved cardiac contractile function at 4 and 12 weeks after transplantation; however, the incidence of ventricular tachycardia was transiently, but significantly, increased when compared to vehicle-treated controls. Collectively, our data demonstrate that allogeneic iPSC-CM transplantation is sufficient to regenerate the infarcted non-human primate heart; however, further research to control post-transplant arrhythmias is necessary.

Deletion of exons 3-9 encompassing a mutational hot spot in the DMD gene presents an asymptomatic phenotype, indicating a target region for multiexon skipping therapy.

Few cases of dystrophinopathy show an asymptomatic phenotype with mutations in the 5' (exons 3-7) hot spot in the Duchenne muscular dystrophy (DMD) gene. Our patient showed increased serum creatine kinase levels at 12 years of age. A muscle biopsy at 15 years of age led to a diagnosis of Becker muscular dystrophy. The patient showed a slight decrease in cardiac function at the age of 21 years and was administered a ß-blocker, but there was no muscle involvement even at the age of 27 years. A deletion of exons 3-9 encompassing a mutational hot spot in the DMD gene was detected, and dystrophin protein expression was ∼15% that of control level. We propose that in-frame deletion of exons 3-9 may produce a functional protein, and that multiexon skipping therapy targeting these exons may be feasible for severe dystrophic patients with a mutation in the 5' hot spot of the DMD gene.

Correlation Between White Matter Lesions and Intelligence Quotient in Patients With Congenital Cytomegalovirus Infection.

BACKGROUND: It is well known that congenital cytomegalovirus infection exhibits white matter and other types of lesions in magnetic resonance imaging (MRI), but little is known on the clinical significance of white matter lesions because they are also present in asymptomatic congenital cytomegalovirus infection. We investigated for relationships among white matter lesions, intelligence quotient, and other neurodevelopmental features. METHODS: Nine children (five boys and four girls; mean age: 87.4 months, range: 63-127 months) with sensorineural hearing loss (five bilateral and four unilateral) had been diagnosed as having congenital cytomegalovirus infection by positive polymerase chain reaction findings of dried umbilical cords. They were evaluated for the presence of autistic features, tested using Wechsler Intelligence Scale for Children-Fourth Edition for intelligence quotient, and underwent brain MRI to measure white matter lesion localization and volume. RESULTS: At the time of MRI examination (mean age: 69.4 months, range: 19-92 months), white matter lesions were detected in eight of nine patients. Five subjects were diagnosed as having autism spectrum disorders. We observed increased white matter lesion volume was associated with lower intelligence quotient scores (R(2) = 0.533, P = 0.026) but not with autism spectrum disorders. CONCLUSIONS: In individuals with congenital cytomegalovirus, an increased white matter lesion volume is associated with lower intelligence quotient scores but not with an increased likelihood of autistic behavior.

Elevation of neuron specific enolase and brain iron deposition on susceptibility-weighted imaging as diagnostic clues for beta-propeller protein-associated neurodegeneration in early childhood: Additional case report and review of the literature.

Beta-propeller protein-associated neurodegeneration (BPAN), also known as static encephalopathy of childhood with neurodegeneration in adulthood (SENDA), is a subtype of neurodegeneration with brain iron accumulation (NBIA). BPAN is caused by mutations in an X-linked gene WDR45 that is involved in autophagy. BPAN is characterized by developmental delay or intellectual disability until adolescence or early adulthood, followed by severe dystonia, parkinsonism, and progressive dementia. Brain magnetic resonance imaging (MRI) shows iron deposition in the bilateral globus pallidus (GP) and substantia nigra (SN). Clinical manifestations and laboratory findings in early childhood are limited. We report a 3-year-old girl with BPAN who presented with severe developmental delay and characteristic facial features. In addition to chronic elevation of serum aspartate transaminase, lactate dehydrogenase, creatine kinase, and soluble interleukin-2 receptor, she had persistent elevation of neuron specific enolase (NSE) in serum and cerebrospinal fluid. MRI using susceptibility-weighted imaging (SWI) demonstrated iron accumulation in the GP and SN bilaterally. Targeted next-generation sequencing identified a de novo splice-site mutation, c.831-1G>C in WDR45, which resulted in aberrant splicing evidenced by reverse transcriptase-PCR. Persistent elevation of NSE and iron deposition on SWI may provide clues for diagnosis of BPAN in early childhood.

Periodic sound-based 6-minute walk test forpatients with Duchenne muscular dystrophy:a preliminary study.

[Purpose] The purpose of this study was to verify if a periodic sound-based 6-minute walk test with the best periodic sound could be used to evaluate physical endurance more precisely than the conventional 6-minute walk test. [Subjects] The subjects were healthy subjects and 6 ambulant patients with Duchenne muscular dystrophy. [Methods] The subjects initially walked for 1 minute to a long-interval metronome sound, and the walking distance was measured. The sound interval was then gradually shortened, and the subjects walked for 1 minute for each of the intervals. The best periodic sound was considered to be the periodic sound used when the subject walked the longest distance in 1 minute, and the process of determining it was referred to as the period shortening walk test. This study administered the 6-minute walk test with the best periodic sound to twenty healthy subjects and 6 ambulant patients with Duchenne muscular dystrophy and compared the walking distance. [Results] The periodic sound-based 6-minute walk test distances in both the healthy subjects and the patients were significantly longer than the conventional 6-minute walk test distances. [Conclusion] The periodic sound-based 6-minute walk test provided a better indication of ambulatory potential in an evaluation of physical endurance than the conventional 6-minute walk test.