SPTLC3 Is Essential for Complex I Activity and Contributes to Ischemic Cardiomyopathy.

BACKGROUND: Dysregulated metabolism of bioactive sphingolipids, including ceramides and sphingosine-1-phosphate, has been implicated in cardiovascular disease, although the specific species, disease contexts, and cellular roles are not completely understood. Sphingolipids are produced by the serine palmitoyltransferase enzyme, canonically composed of 2 subunits, SPTLC1 (serine palmitoyltransferase long chain base subunit 1) and SPTLC2 (serine palmitoyltransferase long chain base subunit 2). Noncanonical sphingolipids are produced by a more recently described subunit, SPTLC3 (serine palmitoyltransferase long chain base subunit 3). METHODS: The noncanonical (d16) and canonical (d18) sphingolipidome profiles in cardiac tissues of patients with end-stage ischemic cardiomyopathy and in mice with ischemic cardiomyopathy were analyzed by targeted lipidomics. Regulation of SPTLC3 by HIF1α under ischemic conditions was determined with chromatin immunoprecipitation. Transcriptomics, lipidomics, metabolomics, echocardiography, mitochondrial electron transport chain, mitochondrial membrane fluidity, and mitochondrial membrane potential were assessed in the cSPTLC3KO transgenic mice we generated. Furthermore, morphological and functional studies were performed on cSPTLC3KO mice subjected to permanent nonreperfused myocardial infarction. RESULTS: Herein, we report that SPTLC3 is induced in both human and mouse models of ischemic cardiomyopathy and leads to production of atypical sphingolipids bearing 16-carbon sphingoid bases, resulting in broad changes in cell sphingolipid composition. This induction is in part attributable to transcriptional regulation by HIF1α under ischemic conditions. Furthermore, cardiomyocyte-specific depletion of SPTLC3 in mice attenuates oxidative stress, fibrosis, and hypertrophy in chronic ischemia, and mice demonstrate improved cardiac function and increased survival along with increased ketone and glucose substrate metabolism utilization. Depletion of SPTLC3 mechanistically alters the membrane environment and subunit composition of mitochondrial complex I of the electron transport chain, decreasing its activity. CONCLUSIONS: Our findings suggest a novel essential role for SPTLC3 in electron transport chain function and a contribution to ischemic injury by regulating complex I activity.

Comprehensive cardiac magnetic resonance T1, T2, and extracellular volume mapping to define Duchenne cardiomyopathy.

BACKGROUND: Cardiomyopathy is the leading cause of death in Duchenne muscular dystrophy (DMD). Cardiac magnetic resonance (CMR) parametric mapping sequences offer insights into disease pathophysiology. We propose a novel approach by leveraging T2 mapping in conjunction with T1 and extracellular volume (ECV) mapping to perform a virtual myocardial biopsy. While previous work has attempted to describe myocardial changes in DMD, our inclusion of T2 mapping enables comprehensive categorization of myocardial tissue characteristics of fibrosis, edema, and fat to better understand the pathological composition of the myocardium with disease progression. METHODS: DMD patients (n = 49; median: 12 years-old) underwent CMR, including T1, T2, and ECV. Categories were defined as normal, isolated high T1 (normal ECV, high T1, normal T2), fibrosis (high ECV, normal or high T1, normal T2), edema (normal or high ECV, normal or high T1, high T2), fat (normal ECV, low T1, high T2) or fibrofatty (high ECV, low T1, high T2). RESULTS: Median left ventricular ejection fraction (LVEF) was 59% with 27% having LVEF < 55%. Those with normal LVEF and no late gadolinium enhancement (37%) were younger in age (10.5 ± 2.6 vs. 15.0 ± 4.3 years-old, p < 0.001). Native T1 was elevated in at least one slice in 82% of patients. Those with high T2 at any slice (27%) were older (p = 0.005) and had lower LVEF (p = 0.005) compared with subjects with normal T2 (73%). The most common myocardial characterization was fibrosis (43%) followed by isolated high T1 (24%). Of the 13 with high T2, ten were categorized as edema, two as fibrofatty, and one as fat. CONCLUSION: CMR parametric mapping sequences offer insights into Duchenne cardiomyopathy pathophysiology, which should drive development of therapeutic interventions aimed at these targets. Myocardial fibrosis is common in DMD. Patients with elevated T2 were older and had lower LVEF. Though fat infiltration was present, the majority of subjects with elevated T2 met criteria for myocardial edema.

Duchenne muscular dystrophy patients: troponin leak in asymptomatic and implications for drug toxicity studies.

BACKGROUND: Cardiomyopathy is the leading cause of death in Duchenne muscular dystrophy (DMD), but studies suggest heart failure biomarkers correlate poorly with cardiomyopathy severity. DMD clinical trials have used troponin I (cTnI) as a biomarker of toxicity, but it is unclear if asymptomatic DMD patients have elevated cTnI. We longitudinally evaluated cTnI, brain natriuretic peptide (BNP), and N-terminal pro-BNP (NT-proBNP) in a DMD cohort. METHODS: DMD patients were prospectively enrolled and followed for 3 years. Serum was drawn at the time of cardiac magnetic resonance (CMR). Normal biomarker values were derived from healthy subjects. Biomarkers were correlated with CMR markers. RESULTS: All subjects were asymptomatic at the time of enrollment. Several DMD subjects had transiently elevated cTnI. Those with elevated cTnI were more likely to have late gadolinium enhancement on baseline CMR. NT-proBNP correlated with indexed left ventricular end diastolic and maximum left atrial volumes. Otherwise, standard cardiac biomarkers did not correlate with CMR markers of cardiomyopathy. CONCLUSIONS: CTnI, BNP, and NT-proBNP do not correlate with CMR assessment of cardiomyopathy progression. A subset of DMD patients have asymptomatic cTnI leak of uncertain clinical significance, though of critical importance if cTnI is used to assess for cardiac toxicity in future drug trials. IMPACT: Asymptomatic patients with Duchenne muscular dystrophy (DMD) exhibit transient troponin I leak. NT-proBNP correlated with indexed left ventricular end diastolic volume and indexed maximum left atrial volume. Other cardiac biomarkers did not correlate with cardiac magnetic resonance (CMR) markers of cardiomyopathy.

Current Practices in Treating Cardiomyopathy and Heart Failure in Duchenne Muscular Dystrophy (DMD): Understanding Care Practices in Order to Optimize DMD Heart Failure Through ACTION.

Cardiac disease has emerged as a leading cause of mortality in Duchenne muscular dystrophy in the current era. This survey sought to identify the diagnostic and therapeutic approach to DMD among pediatric cardiologists in Advanced Cardiac Therapies Improving Outcomes Network. Pediatric cardiology providers within ACTION (a multi-center pediatric heart failure learning network) were surveyed regarding their approaches to cardiac care in DMD. Thirty-one providers from 23 centers responded. Cardiac MRI and Holter monitoring are routinely obtained, but the frequency of use and indications for ordering these tests varied widely. Angiotensin converting enzyme inhibitor and aldosterone antagonist are generally initiated prior to onset of systolic dysfunction, while the indications for initiating beta-blocker therapy vary more widely. Seventeen (55%) providers report their center has placed an implantable cardioverter defibrillator in at least 1 DMD patient, while 11 providers (35%) would not place an ICD for primary prevention in a DMD patient. Twenty-three providers (74%) would consider placement of a ventricular assist device (VAD) as destination therapy (n = 23, 74%) and three providers (10%) would consider a VAD only as bridge to transplant. Five providers (16%) would not consider VAD at their institution. Cardiac diagnostic and therapeutic approaches vary among ACTION centers, with notable variation present regarding the use of advanced therapies (ICD and VAD). The network is currently working to harmonize medical practices and optimize clinical care in an era of rapidly evolving outcomes and cardiac/skeletal muscle therapies.

Non-contrast cardiovascular magnetic resonance detection of myocardial fibrosis in Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) leads to progressive cardiomyopathy. Detection of myocardial fibrosis with late gadolinium enhancement (LGE) by cardiovascular magnetic resonance (CMR) is critical for clinical management. Due to concerns of brain deposition of gadolinium, non-contrast methods for detecting and monitoring myocardial fibrosis would be beneficial. OBJECTIVES: We hypothesized that native T1 mapping and/or circumferential (εcc) and longitudinal (εls) strain can detect myocardial fibrosis. METHODS: 156 CMRs with gadolinium were performed in 66 DMD boys and included: (1) left ventricular ejection fraction (LVEF), (2) LGE, (3) native T1 mapping and myocardial tagging (εcc-tag measured using harmonic phase analysis). LGE was graded as: (1) presence/absence by segment, slice, and globally; (2) global severity from 0 (no LGE) to 4 (severe); (3) percent LGE using full width half maximum (FWHM). εls and εcc measured using feature tracking. Regression models to predict LGE included native T1 and either εcc-tag or εls and εcc measured at each segment, slice, and globally. RESULTS: Mean age and LVEF at first CMR were 14 years and 54%, respectively. Global εls and εcc strongly predicted presence or absence of LGE (OR 2.6 [1.1, 6.0], p = 0.029, and OR 2.3 [1.0, 5.1], p = 0.049, respectively) while global native T1 did not. Global εcc, εls, and native T1 predicted global severity score (OR 2.6 [1.4, 4.8], p = 0.002, OR 2.6 [1.4, 6.0], p = 0.002, and OR 1.8 [1.1, 3.1], p = 0.025, respectively). εls correlated with change in LGE by severity score (n = 33, 3.8 [1.0, 14.2], p = 0.048) and εcc-tag correlated with change in percent LGE by FWHM (n = 34, OR 0.2 [0.1, 0.9], p = 0.01). CONCLUSIONS: Pre-contrast sequences predict presence and severity of LGE, with εls and εcc being more predictive in most models, but there was not an observable advantage over using LVEF as a predictor. Change in LGE was predicted by εls (global severity score) and εcc-tag (FWHM). While statistically significant, our results suggest these sequences are currently not a replacement for LGE and may only have utility in a very limited subset of DMD patients.

Sex-Based Differences in Cardiac Gene Expression and Function in BDNF Val66Met Mice.

Brain-derived neurotrophic factor (BDNF) is a pleiotropic neuronal growth and survival factor that is indispensable in the brain, as well as in multiple other tissues and organs, including the cardiovascular system. In approximately 30% of the general population, BDNF harbors a nonsynonymous single nucleotide polymorphism that may be associated with cardiometabolic disorders, coronary artery disease, and Duchenne muscular dystrophy cardiomyopathy. We recently showed that transgenic mice with the human BDNF rs6265 polymorphism (Val66Met) exhibit altered cardiac function, and that cardiomyocytes isolated from these mice are also less contractile. To identify the underlying mechanisms involved, we compared cardiac function by echocardiography and performed deep sequencing of RNA extracted from whole hearts of all three genotypes (Val/Val, Val/Met, and Met/Met) of both male and female Val66Met mice. We found female-specific cardiac alterations in both heterozygous and homozygous carriers, including increased systolic (26.8%, p = 0.047) and diastolic diameters (14.9%, p = 0.022), increased systolic (57.9%, p = 0.039) and diastolic volumes (32.7%, p = 0.026), and increased stroke volume (25.9%, p = 0.033), with preserved ejection fraction and fractional shortening. Both males and females exhibited lower heart rates, but this change was more pronounced in female mice than in males. Consistent with phenotypic observations, the gene encoding SERCA2 (Atp2a2) was reduced in homozygous Met/Met mice but more profoundly in females compared to males. Enriched functions in females with the Met allele included cardiac hypertrophy in response to stress, with down-regulation of the gene encoding titin (Tcap) and upregulation of BNP (Nppb), in line with altered cardiac functional parameters. Homozygous male mice on the other hand exhibited an inflammatory profile characterized by interferon-γ (IFN-γ)-mediated Th1 immune responses. These results provide evidence for sex-based differences in how the BDNF polymorphism modifies cardiac physiology, including female-specific alterations of cardiac-specific transcripts and male-specific activation of inflammatory targets.

The BDNF rs6265 Polymorphism is a Modifier of Cardiomyocyte Contractility and Dilated Cardiomyopathy.

Brain-derived neurotrophic factor (BDNF) is a neuronal growth and survival factor that harbors cardioprotective qualities that may attenuate dilated cardiomyopathy. In ~30% of the population, BDNF has a common, nonsynonymous single nucleotide polymorphism rs6265 (Val66Met), which might be correlated with increased risk of cardiovascular events. We previously showed that BDNF correlates with better cardiac function in Duchenne muscular dystrophy (DMD) patients. However, the effect of the Val66Met polymorphism on cardiac function has not been determined. The goal of the current study was to determine the effects of rs6265 on BDNF biomarker suitability and DMD cardiac functions more generally. We assessed cardiovascular and skeletal muscle function in human DMD patients segregated by polymorphic allele. We also compared echocardiographic, electrophysiologic, and cardiomyocyte contractility in C57/BL-6 wild-type mice with rs6265 polymorphism and in mdx/mTR (mDMD) mouse model of DMD. In human DMD patients, plasma BDNF levels had a positive correlation with left ventricular function, opposite to that seen in rs6265 carriers. There was also a substantial decrease in skeletal muscle function in carriers compared to the Val homozygotes. Surprisingly, the opposite was true when cardiac function of DMD carriers and non-carriers were compared. On the other hand, Val66Met wild-type mice had only subtle functional differences at baseline but significantly decreased cardiomyocyte contractility. Our results indicate that the Val66Met polymorphism alters myocyte contractility, conferring worse skeletal muscle function but better cardiac function in DMD patients. Moreover, these results suggest a mechanism for the relative preservation of cardiac tissues compared to skeletal muscle in DMD patients and underscores the complexity of BDNF signaling in response to mechanical workload.

Metabolic regulation of Kv channels and cardiac repolarization by Kvß2 subunits.

Voltage-gated potassium (Kv) channels control myocardial repolarization. Pore-forming Kvα proteins associate with intracellular Kvß subunits, which bind pyridine nucleotides with high affinity and differentially regulate channel trafficking, plasmalemmal localization and gating properties. Nevertheless, it is unclear how Kvß subunits regulate myocardial K+ currents and repolarization. Here, we tested the hypothesis that Kvß2 subunits regulate the expression of myocardial Kv channels and confer redox sensitivity to Kv current and cardiac repolarization. Co-immunoprecipitation and in situ proximity ligation showed that in cardiac myocytes, Kvß2 interacts with Kv1.4, Kv1.5, Kv4.2, and Kv4.3. Cardiac myocytes from mice lacking Kcnab2 (Kvß2-/-) had smaller cross sectional areas, reduced sarcolemmal abundance of Kvα binding partners, reduced Ito, IK,slow1, and IK,slow2 densities, and prolonged action potential duration compared with myocytes from wild type mice. These differences in Kvß2-/- mice were associated with greater P wave duration and QT interval in electrocardiograms, and lower ejection fraction, fractional shortening, and left ventricular mass in echocardiographic and morphological assessments. Direct intracellular dialysis with a high NAD(P)H:NAD(P)+ accelerated Kv inactivation in wild type, but not Kvß2-/- myocytes. Furthermore, elevated extracellular levels of lactate increased [NADH]i and prolonged action potential duration in wild type cardiac myocytes and perfused wild type, but not Kvß2-/-, hearts. Taken together, these results suggest that Kvß2 regulates myocardial electrical activity by supporting the functional expression of proteins that generate Ito and IK,slow, and imparting redox and metabolic sensitivity to Kv channels, thereby coupling cardiac repolarization to myocyte metabolism.

Tissue characterisation and myocardial mechanics using cardiac MRI in children with hypertrophic cardiomyopathy.

INTRODUCTION: Distinguishing between hypertrophic cardiomyopathy and other causes ofleft ventricular hypertrophy can be difficult in children. We hypothesised that cardiac MRI T1 mapping could improve diagnosis of paediatric hypertrophic cardiomyopathy and that measures of myocardial function would correlate with T1 times and extracellular volume fraction. METHODS: Thirty patients with hypertrophic cardiomyopathy completed MRI with tissue tagging, T1-mapping, and late gadolinium enhancement. Left ventricular circumferential strain was calculated from tagged images. T1, partition coefficient, and synthetic extracellular volume were measured at base, mid, apex, and thickest area of myocardial hypertrophy. MRI measures compared to cohort of 19 healthy children and young adults. Mann-Whitney U, Spearman's rho, and multivariable logistic regression were used for statistical analysis. RESULTS: Hypertrophic cardiomyopathy patients had increased left ventricular ejection fraction and indexed mass. Hypertrophic cardiomyopathy patients had decreased global strain and increased native T1 (-14.3% interquartile range [-16.0, -12.1] versus -17.3% [-19.0, -15.7], p < 0.001 and 1015 ms [991, 1026] versus 990 ms [972, 1001], p = 0.019). Partition coefficient and synthetic extracellular volume were not increased in hypertrophic cardiomyopathy. Global native T1 correlated inversely with ejection fraction (ρ = -0.63, p = 0.002) and directly with global strain (ρ = 0.51, p = 0.019). A logistic regression model using ejection fraction and native T1 distinguished between hypertrophic cardiomyopathy and control with an area under the receiver operating characteristic curve of 0.91. CONCLUSION: In this cohort of paediatric hypertrophic cardiomyopathy, strain was decreased and native T1 was increased compared with controls. Native T1 correlated with both ejection fraction and strain, and a model using native T1 and ejection fraction differentiated patients with and without hypertrophic cardiomyopathy.

Synthetic hematocrit derived from the longitudinal relaxation of blood can lead to clinically significant errors in measurement of extracellular volume fraction in pediatric and young adult patients.

BACKGROUND: Extracellular volume fraction (ECV) is altered in pathological cardiac remodeling and predicts death and arrhythmia. ECV can be quantified using cardiovascular magnetic resonance (CMR) T1 mapping but calculation requires a measured hematocrit (Hct). The longitudinal relaxation of blood has been used in adults to generate a synthetic Hct (estimate of true Hct) but has not been validated in pediatric populations. METHODS: One hundred fourteen children and young adults underwent a total of 163 CMRs with T1 mapping. The majority of subjects had a measured Hct the same day (N = 146). Native and post-contrast T1 were determined in blood pool, septum, and free wall of mid-LV, avoiding areas of late gadolinium enhancement. Synthetic Hct and ECV were calculated and intraclass correlation coefficient (ICC) and linear regression were used to compare measured and synthetic values. RESULTS: The mean age was 16.4 ± 6.4 years and mean left ventricular ejection fraction was 59% ± 9%. The mean measured Hct was 41.8 ± 3.0% compared to the mean synthetic Hct of 43.2% ± 2.9% (p < 0.001, ICC 0.46 [0.27, 0.52]) with the previously published model and 41.8% ± 1.4% (p < 0.001, ICC 0.28 [0.13, 0. 42]) with the locally-derived model. Mean measured mid-free wall ECV was 30.5% ± 4.8% and mean synthetic mid-free wall ECV of local model was 29.7% ± 4.6% (p < 0.001, ICC 0.93 [0.91, 0.95]). Correlations were not affected by heart rate and did not significantly differ in subpopulation analysis. While the ICC was strong, differences between measured and synthetic ECV ranged from -8.4% to 4.3% in the septum and -12.6% to 15.8% in the free wall. Using our laboratory's normal cut-off of 28.5%, 59 patients (37%) were miscategorized (53 false negatives, 6 false positives) with published model ECV. The local model had 37 miscategorizations (20 false negatives, 17 false positives), significantly fewer but still a substantial number (23%). CONCLUSIONS: Our data suggest that use of synthetic Hct for the calculation of ECV results in miscategorization of individual patients. This difference may be less significant once synthetic ECV is calculated and averaged over a large research cohort, making it potentially useful as a research tool. However, we recommend formal measurement of Hct in children and young adults for clinical CMRs.

Rate of Change in Cardiac Magnetic Resonance Imaging Measures Is Associated With Death in Duchenne Muscular Dystrophy.

BACKGROUND: Cardiovascular disease is the leading cause of death among patients with Duchenne muscular dystrophy (DMD). Identifying patients at risk of early death could allow for increased monitoring and more intensive therapy. Measures that associate with death could serve as surrogate outcomes in clinical trials. METHODS AND RESULTS: Duchenne muscular dystrophy subjects prospectively enrolled in observational studies were included. Models using generalized least squares were used to assess the difference of cardiac magnetic resonance measurements between deceased and alive subjects. A total of 63 participants underwent multiple cardiac magnetic resonance imaging and were included in the analyses. Twelve subjects (19.1%) died over a median follow-up of 5 years (interquartile range, 3.1-7.0). Rate of decline in left ventricular ejection fraction was faster in deceased than alive subjects (P<0.0001). Rate of increase in indexed left ventricular end-diastolic (P=0.0132) and systolic (P<0.0001) volumes were higher in deceased subjects. Faster worsening in midcircumferential strain was seen in deceased subjects (P=0.049) while no difference in global circumferential strain was seen. The rate of increase in late gadolinium enhancement, base T1, and mid T1 did not differ between groups. CONCLUSIONS: Duchenne muscular dystrophy death is associated with the rate of change in left ventricular ejection fraction, midcircumferential strain, and ventricular volumes. Aggressive medical therapy to decrease the rate of progression may improve the mortality rate in this population. A decrease in the rate of progression may serve as a valid surrogate outcome for therapeutic trials.

Right-ventricular global longitudinal strain may predict neo-aortic arch obstruction after Norwood/Sano procedure in children with hypoplastic left heart syndrome.

Neo-aortic arch obstruction (NAAO) is a common complication following the Norwood/Sano procedure (NP) for hypoplastic left heart syndrome (HLHS) and is associated with increased morbidity and mortality. However, there is currently no objective method for predicting which patients will develop NAAO. This study was designed to test the hypothesis that hemodynamic changes from development of NAAO after NP in patients with HLHS will lead to changes in myocardial dynamics that could be detected before clinical symptoms develop with strain analysis using velocity vector imaging. Patients with HLHS who had at least one cardiac catheterization after NP were identified retrospectively. Strain analysis was performed on all echocardiograms preceding the first catheterization and any subsequent catheterization performed for intervention on NAAO. Twelve patients developed NAAO and 30 patients never developed NAAO. Right ventricular strain was worse in the group that developed NAAO (-6.2 vs. -8.6 %, p = 0.040) at a median of 59 days prior to diagnosis of NAAO. Those patients that developed NAAO following NP were significantly younger at the time of first catheterization than those that did not develop NAAO (92 ± 50 vs. 140 ± 36 days, p = 0.001). This study demonstrates that right ventricular GLS is abnormal in HLHS patients following NP and worsening right ventricular strain may be predictive of the future development of NAAO.

Cardiovascular Measures of All-Cause Mortality in Duchenne Muscular Dystrophy.

BACKGROUND: Cardiopulmonary failure is the leading cause of death in Duchenne muscular dystrophy (DMD). Research into DMD-specific cardiovascular therapies is ongoing, but there are no Food and Drug Administration-approved cardiac end points. To adequately power a therapeutic trial, appropriate end points must be chosen and the rate of change for these end points reported. The objective of this study was to evaluate rate of change for cardiac magnetic resonance and blood biomarkers and to determine which measures associate with all-cause mortality in DMD. METHODS: Seventy-eight DMD subjects underwent 211 cardiac magnetic resonance studies analyzed for left ventricular (LV) ejection fraction, indexed LV end diastolic and systolic volumes, circumferential strain, late gadolinium enhancement presence and severity (global severity score, and full width half maximum), native T1 mapping, T2 mapping, and extracellular volume. Blood samples were analyzed for BNP (brain natriuretic peptide), NT-proBNP (N-terminal pro-B-type natriuretic peptide), and troponin I. Cox proportional hazard regression modeling was performed with all-cause mortality as the outcome. RESULTS: Fifteen subjects (19%) died. LV ejection fraction, indexed end systolic volumes, global severity score, and full width half maximum worsened at 1 and 2 years while circumferential strain and indexed LV end diastolic volumes worsened at 2 years. LV ejection fraction, indexed LV end diastolic and systolic volumes, late gadolinium enhancement full width half maximum, and circumferential strain associated with all-cause mortality (P<0.05). NT-proBNP was the only blood biomarker that associated with all-cause mortality (P<0.05). CONCLUSIONS: LV ejection fraction, indexed LV volumes, circumferential strain, late gadolinium enhancement full width half maximum, and NT-proBNP are associated with all-cause mortality in DMD and may be the best end points for use in cardiovascular therapeutic trials. We also report change over time of cardiac magnetic resonance and blood biomarkers.

Cardiovascular Disease in Duchenne Muscular Dystrophy: Overview and Insight Into Novel Therapeutic Targets.

Duchenne muscular dystrophy (DMD) is a devastating disease affecting approximately 1 in every 3,500 male births worldwide. Multiple mutations in the dystrophin gene have been implicated as underlying causes of DMD. However, there remains no cure for patients with DMD, and cardiomyopathy has become the most common cause of death in the affected population. Extensive research is under way investigating molecular mechanisms that highlight potential therapeutic targets for the development of pharmacotherapy for DMD cardiomyopathy. In this paper, the authors perform a literature review reporting on recent ongoing efforts to identify novel therapeutic strategies to reduce, prevent, or reverse progression of cardiac dysfunction in DMD.

Improving Access and Guideline Adherence in Pulmonary Care in Patients With Duchenne Muscular Dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a devastating, progressive neuromuscular disease that results in cardiopulmonary failure and death. In 2018, the DMD Care Considerations guidelines were updated to improve the multidisciplinary approach to care and promote early respiratory management. We sought to evaluate the impact of a multidisciplinary clinic on access to pulmonary care and adherence to respiratory care guidelines. METHODS: Utilizing retrospective data, we assessed for pulmonary care between 2016-2019 and congruence with guidelines from March 2018-February 2019. Using a standardized visit protocol, subjects were monitored for adherence to pulmonary function testing (PFT) and polysomnography (PSG) recommendations. RESULTS: Of the 84 subjects with DMD, only 51.2% had prior pulmonary involvement, and approximately one-third were seen in the year prior to clinic onset. Only 23% of subjects with a pulmonary referral completed this visit. After clinic initiation, the average age of a subject's first pulmonary contact decreased from 11.8 y to 7.9 y (P < .001), and 45% of the 77 unique clinic subjects had no previous pulmonary encounter. Adherence to PFT guidelines increased in both ambulatory (8.7% to 86.1%) and non-ambulatory subjects (25.9% to 90.1%). Approximately 79% of subjects seen in clinic either completed or had an order for PSG in the last 12 months. CONCLUSIONS: Development of a multispecialty clinic expanded access to pulmonary care and evaluation in subjects with DMD. Continued care in this clinic will allow a better understanding of barriers to access and the opportunity to monitor long-term pulmonary health.

Regulation of swelling-activated Cl(-) current by angiotensin II signalling and NADPH oxidase in rabbit ventricle.

AIMS: We assessed whether hypoosmotic swelling of cardiac myocytes activates volume-sensitive Cl(-) current (I Cl,swell) via the angiotensin II (AngII)-reactive oxygen species (ROS) signalling cascade. The AngII-ROS pathway previously was shown to elicit I(Cl,swell) upon mechanical stretch of beta(1D) integrin. Integrin stretch and osmotic swelling are, however, distinct stimuli. For example, blocking Src kinases stimulates swelling-induced but inhibits stretch-induced I Cl,swell. METHODS AND RESULTS: I Cl,swell was measured in rabbit ventricular myocytes by whole-cell voltage clamp. Swelling-induced I Cl,swell was completely blocked by losartan and eprosartan, AngII type I receptor (AT1) antagonists. AT1 stimulation transactivates epidermal growth factor receptor (EGFR) kinase. Blockade of EGFR kinase with AG1478 abolished both I Cl,swell and AngII-induced Cl(-) current, whereas exogenous EGF evoked a Cl(-) current that was suppressed by osmotic shrinkage. Phosphatidylinositol 3-kinase (PI-3K) is downstream of EGFR kinase, and PI-3K inhibitors LY294002 and wortmannin blocked I Cl,swell. Ultimately, AngII signals via NADPH oxidase (NOX) and superoxide anion, O2*. NOX inhibitors, diphenyleneiodonium, apocynin and gp91ds-tat, eliminated I Cl,swell, whereas scramb-tat, an inactive gp91ds-tat analogue, was ineffective. O2* rapidly dismutates to H2O2. Consistent with H2O2 being a downstream effector, catalase inhibited I Cl,swell, and exogenous H2O2 overcame suppression of I Cl,swell by AT1 receptor, EGFR kinase, and PI-3K blockers. H2O2-induced current was not blocked by osmotic shrinkage, however. CONCLUSION: Activation of I Cl,swell by osmotic swelling is controlled by the AngII-ROS cascade, the same pathway previously implicated in I Cl,swell activation by integrin stretch. This in part explains why I Cl,swell is persistently activated in several models of cardiac disease.

A high resolution structure of the putative hinge region in M2 channel-lining segments of the nicotinic acetylcholine receptor.

Transmembrane peptide helices play key roles in signal transduction across cell membranes, yet little is known about their high-resolution structure or the role membrane composition plays in their association, structure, dynamics and ultimately their performance. Using magic angle spinning (MAS) homonuclear dipolar recoupling experiments, the backbone structure at positions L10, L11, and A12 of the M2 ion channel peptide was determined in two lipid systems. Their measurements are in agreement with M2 forming transmembrane helices, but the torsion angles vary considerably from common alpha-helical values. These measurements show remarkable agreement with a previous computational model of M2 peptides forming a pore domain in which their helices are kinked near the central leucine, L11 [R. Sankararamakrishnan, C. Adcock, M.S.P. Sansom, The pore domain of the nicotinic acetylcholine receptor: Molecular modeling, pore dimensions, and electrostatics, Biophys. J. 71 (1996) 1659-1671]. The generation of high resolution data for transmembrane helices is of critical importance in refining structures for membrane protein and developing models of helix packing interactions.

Increased Number of Circulating CD8/CD26 T Cells in the Blood of Duchenne Muscular Dystrophy Patients Is Associated with Augmented Binding of Adenosine Deaminase and Higher Muscular Strength Scores.

Duchenne muscular dystrophy (DMD) is an X-linked disorder that leads to cardiac and skeletal myopathy. The complex immune activation in boys with DMD is incompletely understood. To better understand the contribution of the immune system into the progression of DMD, we performed a systematic characterization of immune cell subpopulations obtained from peripheral blood of DMD subjects and control donors. We found that the number of CD8 cells expressing CD26 (also known as adenosine deaminase complexing protein 2) was increased in DMD subjects compared to control. No differences, however, were found in the levels of circulating factors associated with pro-inflammatory activation of CD8/CD26 cells, such as tumor necrosis factor-α (TNFα), granzyme B, and interferon-γ (IFNγ). The number of CD8/CD26 cells correlated directly with quantitative muscle testing (QMT) in DMD subjects. Since CD26 mediates binding of adenosine deaminase (ADA) to the T cell surface, we tested ADA-binding capacity of CD8/CD26 cells and the activity of bound ADA. We found that mononuclear cells (MNC) obtained from DMD subjects with an increased number of CD8/CD26 T cells had a greater capacity to bind ADA. In addition, these MNC demonstrated increased hydrolytic deamination of adenosine to inosine. Altogether, our data demonstrated that (1) an increased number of circulating CD8/CD26 T cells is associated with preservation of muscle strength in DMD subjects, and (2) CD8/CD26 T cells from DMD subjects mediated degradation of adenosine by adenosine deaminase. These results support a role for T cells in slowing the decline in skeletal muscle function, and a need for further investigation into contribution of CD8/CD26 T cells in the regulation of chronic inflammation associated with DMD.

Predicting economic and medical outcomes based on risk adjustment for congenital heart surgery classification of pediatric cardiovascular surgical admissions.

The Risk Adjustment for Congenital Heart Surgery (RACHS-1) classification is an established method for predicting mortality for congenital heart disease surgery. It is unknown if this extends to the cost of hospitalization or if differences in economic and medical outcomes exist in certain subpopulations. Using data obtained from the University HealthSystem Consortium, we examined inpatient resource use by patients with International Classification of Diseases, Ninth Revision, procedure codes representative of RACHS-1 classifications 1 through 5 and 6 from 2006 to 2012. A total of 15,453 pediatric congenital heart disease surgical admissions were analyzed, with overall mortality of 4.5% (n = 689). As RACHS-1 classification increased, the total cost of hospitalization, hospital charges, total length of stay, length of intensive care unit stay, and mortality increased. Even when controlled for RACHS-1 classification, black patients (n = 2034) had higher total costs ($96,884 ± $3,392, p = 0.003), higher charges ($318,313 ± $12,018, p <0.001), and longer length of stay (20.4 ± 0.7 days, p <0.001) compared with white patients ($85,396 ± $1,382, $285,622 ± $5,090, and 18.0 ± 0.3 days, respectively). Hispanic patients had similarly disparate outcomes ($104,292 ± $2,759, $351,371 ± $10,627, and 23.0 ± 0.6 days, respectively) and also spent longer in the intensive care unit (14.9 ± 0.5 days, p <0.001). In conclusion, medical and economic measures increased predictably with increased procedure risk, and admissions for black and Hispanic patients were longer and more expensive than those of their white counterparts but without increased mortality.

Head and neck vessel size by angiography predicts neo-aortic arch obstruction after Norwood/Sano operation for hypoplastic left heart syndrome.

OBJECTIVES: To identify and predict neo-aortic arch obstruction (NAAO) in children after Norwood/Sano operation (NO) for hypoplastic left heart syndrome (HLHS). BACKGROUND: NAAO is associated with morbidity and mortality after NO for HLHS and no objective measure has predicted the initial occurrence of NAAO. Computational flow models of aortic coarctation demonstrate increased wall shear stress (WSS) in vessels proximal to the coarctation segment, which we believe also occurs with NAAO. These vessels respond by increasing their luminal diameter to maintain normal WSS. We hypothesized that the relative increase in diameters of head and neck vessels to the isthmus, as measured by angiography, would identify hemodynamically significant NAAO and predict future NAAO. METHODS: Retrospective review of patients with HLHS and at least one catheterization with aortic angiography after NO. Diameters of head and neck vessels were totaled and divided by the isthmus diameter to give a head and neck index (HNI), which was compared to coarctation index (CI) for identifying and predicting future NAAO. RESULTS: Forty-four patients were identified, 17 with and 27 without NAAO. Receiver operator characteristic analysis using a value for CI ≤0.5 showed a sensitivity of 47% and specificity of 89%. For HNI, a value >2.65 gave a sensitivity of 77% and specificity of 93%. Three patients who developed NAAO after their initial catheterization had CI >0.5, but abnormally high HNI >2.65. CONCLUSIONS: HNI is a more robust indicator of hemodynamically significant NAAO than CI and may predict its future occurrence after NO for HLHS.