Cardiomyocyte-produced miR-339-5p mediates pathology in Duchenne muscular dystrophy cardiomyopathy.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized by severe, progressive muscle wasting. Cardiomyopathy has emerged as a leading cause of death in patients with DMD. The mechanisms contributing to DMD cardiac disease remain under investigation and specific therapies available are lacking. Our prior work has shown that DMD-iPSC-derived cardiomyocytes (DMD-iCMs) are vulnerable to oxidative stress injury and chronic exposure to DMD-secreted exosomes impaired the cell's ability to protect against stress. In this study, we sought to examine a mechanism by which DMD cardiac exosomes impair cellular response through altering important stress-responsive genes in the recipient cells. Here, we report that DMD-iCMs secrete exosomes containing altered microRNA (miR) profiles in comparison to healthy controls. In particular, miR-339-5p was upregulated in DMD-iCMs, DMD exosomes and mdx mouse cardiac tissue. Restoring dystrophin in DMD-iCMs improved the cellular response to stress and was associated with downregulation of miR-339-5p, suggesting that it is disease-specific. Knockdown of miR-339-5p was associated with increased expression of MDM2, GSK3A and MAP2K3, which are genes involved in important stress-responsive signaling pathways. Finally, knockdown of miR-339-5p led to mitochondrial protection and a reduction in cell death in DMD-iCMs, indicating miR-339-5p is involved in direct modulation of stress-responsiveness. Together, these findings identify a potential mechanism by which exosomal miR-339-5p may be modulating cell signaling pathways that are important for robust stress responses. Additionally, these exosomal miRs may provide important disease-specific targets for future therapeutic advancements for the management and diagnosis of DMD cardiomyopathy.

Cardioprotective effect of nicorandil on isoproterenol induced cardiomyopathy in the Mdx mouse model.

BACKGROUND: Duchenne muscular dystrophy (DMD) associated cardiomyopathy is a major cause of morbidity and mortality. In an in vitro DMD cardiomyocyte model, nicorandil reversed stress-induced cell injury through multiple pathways implicated in DMD. We aimed to test the efficacy of nicorandil on the progression of cardiomyopathy in mdx mice following a 10-day treatment protocol. METHODS: A subset of mdx mice was subjected to low-dose isoproterenol injections over 5 days to induce a cardiac phenotype and treated with vehicle or nicorandil for 10 days. Baseline and day 10 echocardiograms were obtained to assess cardiac function. At 10 days, cardiac tissue was harvested for further analysis, which included histologic analysis and assessment of oxidative stress. Paired student's t test was used for in group comparison, and ANOVA was used for multiple group comparisons. RESULTS: Compared to vehicle treated mice, isoproterenol decreased ejection fraction and fractional shortening on echocardiogram. Nicorandil prevented isoproterenol induced cardiac dysfunction. Isoproterenol increased cardiac fibrosis, which nicorandil prevented. Isoproterenol increased gene expression of NADPH oxidase, which decreased to baseline with nicorandil treatment. Superoxide dismutase 2 protein expression increased in those treated with nicorandil, and xanthine oxidase activity decreased in mice treated with nicorandil during isoproterenol stress compared to all other groups. CONCLUSIONS: In conclusion, nicorandil is cardioprotective in mdx mice and warrants continued investigation as a therapy for DMD associated cardiomyopathy.

Influence of microRNAs and exosomes in muscle health and diseases.

microRNAs are short, (18-22 nt) non-coding RNAs involved in important cellular processes due to their ability to regulate gene expression at the post-transcriptional level. Exosomes are small (50-200 nm) extracellular vesicles, naturally secreted from a variety of living cells and are believed to mediate cell-cell communication through multiple mechanisms, including uptake in destination cells. Circulating microRNAs and exosome-derived microRNAs can have key roles in regulating muscle cell development and differentiation. Several microRNAs are highly expressed in muscle and their regulation is important for myocyte homeostasis. Changes in muscle associated microRNA expression are associated with muscular diseases including muscular dystrophies, inflammatory myopathies, and congenital myopathies. In this review, we aim to highlight the biology of microRNAs and exosomes as well as their roles in muscle health and diseases. We also discuss the potential crosstalk between skeletal and cardiac muscle through exosomes and their contents.

Epitope Mapping of SERCA2a Identifies an Antigenic Determinant That Induces Mainly Atrial Myocarditis in A/J Mice.

Sarcoplasmic/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA)2a, a critical regulator of calcium homeostasis, is known to be decreased in heart failure. Patients with myocarditis or dilated cardiomyopathy develop autoantibodies to SERCA2a suggesting that they may have pathogenetic significance. In this report, we describe epitope mapping analysis of SERCA2a in A/J mice that leads us to make five observations: 1) SERCA2a contains multiple T cell epitopes that induce varying degrees of myocarditis. One epitope, SERCA2a 971-990, induces widespread atrial inflammation without affecting noncardiac tissues; the cardiac abnormalities could be noninvasively captured by echocardiography, electrocardiography, and magnetic resonance microscopy imaging. 2) SERCA2a 971-990-induced disease was associated with the induction of CD4 T cell responses and the epitope preferentially binds MHC class II/IAk rather than IEk By creating IAk/and IEk/SERCA2a 971-990 dextramers, the T cell responses were determined by flow cytometry to be Ag specific. 3) SERCA2a 971-990-sensitized T cells produce both Th1 and Th17 cytokines. 4) Animals immunized with SERCA2a 971-990 showed Ag-specific Abs with enhanced production of IgG2a and IgG2b isotypes, suggesting that SERCA2a 971-990 can potentially act as a common epitope for both T cells and B cells. 5) Finally, SERCA2a 971-990-sensitized T cells were able to transfer disease to naive recipients. Together, these data indicate that SERCA2a is a critical autoantigen in the mediation of atrial inflammation in mice and that our model may be helpful to study the inflammatory events that underlie the development of conditions such as atrial fibrillation in humans.

Identifying Phenogroups in patients with subclinical diastolic dysfunction using unsupervised statistical learning.

BACKGROUND: Subclinical diastolic dysfunction is a precursor for developing heart failure with preserved ejection fraction (HFpEF); yet not all patients progress to HFpEF. Our objective was to evaluate clinical and echocardiographic variables to identify patients who develop HFpEF. METHODS: Clinical, laboratory, and echocardiographic data were retrospectively collected for 81 patients without HF and 81 matched patients with HFpEF at the time of first documentation of subclinical diastolic dysfunction. Density-based clustering or hierarchical clustering to group patients was based on 65 total variables including 19 categorical and 46 numerical variables. Logistic regression analysis was conducted on the entire study population as well as each individual cluster to identify independent predictors of HFpEF. RESULTS: Unsupervised clustering identified 3 subgroups which differed in gender composition, severity of cardiac hypertrophy and aortic stenosis, NT-proBNP, percentage of patients who progressed to HFpEF, and timing of disease progression from diastolic dysfunction to HFpEF to death. Clusters that had higher percentages of women had progressively milder cardiac hypertrophy, less severe aortic stenosis, lower NT-proBNP, were diagnosed at an older age with HFpEF, and survived to an older age. Independent predictors of HFpEF for the entire cohort included diabetes, chronic kidney disease, atrial fibrillation, and diuretic use, with additional predictive variables found for each cluster. CONCLUSIONS: Cluster analysis can identify phenotypically distinct subgroups of patients with diastolic dysfunction. Clusters differ in HFpEF and mortality outcome. In addition, the variables that correlate with and predict HFpEF outcome differ among clusters.

Mapping genetic modifiers of radiation-induced cardiotoxicity to rat chromosome 3.

Radiation therapy is used in ~50% of cancer patients to reduce the risk of recurrence and in some cases improve survival. Despite these benefits, doses can be limited by toxicity in multiple organs, including the heart. The underlying causes and biomarkers of radiation-induced cardiotoxicity are currently unknown, prompting the need for experimental models with inherent differences in sensitivity and resistance to the development of radiation-induced cardiotoxicity. We have identified the parental SS (Dahl salt-sensitive/Mcwi) rat strain to be a highly-sensitized model of radiation-induced cardiotoxicity. In comparison, substitution of rat chromosome 3 from the resistant BN (Brown Norway) rat strain onto the SS background (SS-3BN consomic) significantly attenuated radiation-induced cardiotoxicity. SS-3BN rats had less radiation-induced cardiotoxicity than SS rats, as measured by survival, pleural and pericardial effusions, echocardiogram parameters, and histological damage. Mast cells, previously shown to have predominantly protective roles in radiation-induced cardiotoxicity, were increased in the more resistant SS-3BN hearts postradiation. RNA sequencing from SS and SS-3BN hearts at 1 wk postradiation revealed 5,098 differentially expressed candidate genes across the transcriptome and 350 differentially expressed genes on rat chromosome 3, which coincided with enrichment of multiple pathways, including mitochondrial dysfunction, sirtuin signaling, and ubiquitination. Upstream regulators of enriched pathways included the oxidative stress modulating transcription factor, Nrf2, which is located on rat chromosome 3. Nrf2 target genes were also differentially expressed in the SS vs. SS-3BN consomic hearts postradiation. Collectively, these data confirm the existence of heritable modifiers in radiation-induced cardiotoxicity and provide multiple biomarkers, pathways, and candidate genes for future analyses. NEW & NOTEWORTHY This novel study reveals that heritable genetic factors have the potential to modify normal tissue sensitivity to radiation. Gene variant(s) on rat chromosome 3 can contribute to enhanced cardiotoxicity displayed in the SS rats vs. the BN and SS-3BN consomic rats. Identifying genes that lead to understanding the mechanisms of radiation-induced cardiotoxicity represents a novel method to personalize radiation treatment, as well as predict the development of radiation-induced cardiotoxicity.

Systemic AAV8-Mediated Gene Therapy Drives Whole-Body Correction of Myotubular Myopathy in Dogs.

X-linked myotubular myopathy (XLMTM) results from MTM1 gene mutations and myotubularin deficiency. Most XLMTM patients develop severe muscle weakness leading to respiratory failure and death, typically within 2 years of age. Our objective was to evaluate the efficacy and safety of systemic gene therapy in the p.N155K canine model of XLMTM by performing a dose escalation study. A recombinant adeno-associated virus serotype 8 (rAAV8) vector expressing canine myotubularin (cMTM1) under the muscle-specific desmin promoter (rAAV8-cMTM1) was administered by simple peripheral venous infusion in XLMTM dogs at 10 weeks of age, when signs of the disease are already present. A comprehensive analysis of survival, limb strength, gait, respiratory function, neurological assessment, histology, vector biodistribution, transgene expression, and immune response was performed over a 9-month study period. Results indicate that systemic gene therapy was well tolerated, prolonged lifespan, and corrected the skeletal musculature throughout the body in a dose-dependent manner, defining an efficacious dose in this large-animal model of the disease. These results support the development of gene therapy clinical trials for XLMTM.

Aortic stiffening precedes onset of heart failure with preserved ejection fraction in patients with asymptomatic diastolic dysfunction.

BACKGROUND: Identifying which patients with diastolic dysfunction will progress to heart failure with preserved ejection fraction (HFpEF) remains challenging. The goal of this study is to determine whether increased vascular stiffness as identified on 2D transthoracic echocardiography (TTE) serves as a biomarker for the development of HFpEF in patients with diastolic dysfunction. METHODS: The study design is a matched retrospective case-control study. Subjects with diastolic dysfunction were divided into two groups based on whether they had a clinical diagnosis of HFpEF. The two groups were matched based on age, gender, race and body surface area, resulting in 77 matched pairs (n = 154). Data from the first TTE that documented diastolic dysfunction prior to the development of HFpEF was extracted along with baseline demographic and clinical data. Indices of vascular stiffness were measured and compared. A sub-group analysis was performed to compare diabetic subjects in Group 1 (n = 43) to those in Group 2 (n = 21). RESULTS: Group 1 had significantly decreased aortic distensibility as measured on the initial TTE when compared to Group 2 (1.9 ± 1.0 vs. 2.8 ± 1.8 cm2dyne-110-3, p = 0.01). In the diabetic subset, Group 1 had significantly less aortic strain (6.9 ± 3.3 vs. 9.7 ± 5.6%, p = 0.02) and aortic distensibility (1.8 ± 1.0 vs. 3.5 ± 2.6 cm2dyne-110-3, p = 0.02) compared to Group 2. Other indices of vascular stiffness did not differ significantly between groups. CONCLUSIONS: This study demonstrates that increased proximal aortic stiffness is associated with the development of HFpEF in patients with asymptomatic diastolic dysfunction. Larger prospective studies are needed to further investigate this relationship.

Combining patient proteomics and in vitro cardiomyocyte phenotype testing to identify potential mediators of heart failure with preserved ejection fraction.

BACKGROUND: Heart failure with ejection fraction (HFpEF) is a syndrome resulting from several co-morbidities in which specific mediators are unknown. The platelet proteome responds to disease processes. We hypothesize that the platelet proteome will change composition in patients with HFpEF and may uncover mediators of the syndrome. METHODS AND RESULTS: Proteomic changes were assessed in platelets from hospitalized subjects with symptoms of HFpEF (n = 9), the same subjects several weeks later without symptoms (n = 7) and control subjects (n = 8). Mass spectrometry identified 6102 proteins with five scans with peptide probabilities of ≥0.85. Of the 6102 proteins, 165 were present only in symptomatic subjects, 78 were only found in outpatient subjects and 157 proteins were unique to the control group. The S100A8 protein was identified consistently in HFpEF samples when compared with controls. We validated the fining that plasma S100A8 levels are increased in subjects with HFpEF (654 ± 391) compared to controls (352 ± 204) in an external cohort (p = 0.002). Recombinant S100A8 had direct effects on the electrophysiological and calcium handling profile in human induced pluripotent stem cell-derived cardiomyocytes. CONCLUSIONS: Platelets may harbor proteins associated with HFpEF. S100A8 is present in the platelets of subjects with HFpEF and increased in the plasma of the same subjects. We further established a bedside-to-bench translational system that can be utilized as a secondary screen to ascertain whether the biomarkers may be an associated finding or causal to the disease process. S100A8 has been linked with other cardiovascular disease such as atherosclerosis and risk for myocardial infarction, stroke, or death. This is the first report on association of S100A8 with HFpEF.

A GIPR antagonist conjugated to GLP-1 analogues promotes weight loss with improved metabolic parameters in preclinical and phase 1 settings.

Obesity is a major public health crisis. Multi-specific peptides have emerged as promising therapeutic strategies for clinical weight loss. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are endogenous incretins that regulate weight through their receptors (R). AMG 133 (maridebart cafraglutide) is a bispecific molecule engineered by conjugating a fully human monoclonal anti-human GIPR antagonist antibody to two GLP-1 analogue agonist peptides using amino acid linkers. Here, we confirm the GIPR antagonist and GLP-1R agonist activities in cell-based systems and report the ability of AMG 133 to reduce body weight and improve metabolic markers in male obese mice and cynomolgus monkeys. In a phase 1, randomized, double-blind, placebo-controlled clinical study in participants with obesity ( NCT04478708 ), AMG 133 had an acceptable safety and tolerability profile along with pronounced dose-dependent weight loss. In the multiple ascending dose cohorts, weight loss was maintained for up to 150 days after the last dose. These findings support continued clinical evaluation of AMG 133.

Nerve growth factor reduces myocardial ischemia/reperfusion injury in rat hearts.

BACKGROUND: Nerve growth factor (NGF) is a neurotrophin that supports the survival and differentiation of sympathetic neurons, and its increased expression after myocardial infarct was correlated with cardiac sympathetic hyperinnervation and arrhythmias. However, it is unclear whether NGF protects the heart during infarct. In this study, we sought to address this issue in rat heart exposed to ischemia/reperfusion injury (IRI). METHODS: NGF was administered intravenously (IV), 15 min before ischemia, at different concentrations in the absence or presence of inhibitors of phosphatidylinositol-3 kinase (PI3K) or nitric oxide synthase (NOS) in different groups of rats (n=6) with left coronary occlusion for 30 min followed by 120-min reperfusion. The area at risk and infarct to risk ratios were determined from sections stained with 1% 2,3,5-triphenylterazolium chloride. RESULTS: NGF treatment at doses of 0.015-15 µg/kg, with an optimal dose of 0.15 µg/kg given IV before ischemia, reduced the infarct size from about 60% at the area of risk to about 25%, indicating cardioprotection by about 60%. The infarct-sparing effects of NGF were partially abolished by the inhibition of PI3K and NOS using wortmannin and N(G)-monomethyl-l-arginine, respectively. CONCLUSIONS: We have demonstrated for the first time that NGF attenuates myocardial infarct damage in an in vivo rat model of myocardial regional IRI. This cardioprotective effect is proposed to be related to the activities of PI3K and NOS. This suggests that NGF has a potential therapeutic role in the treatment of IRI.

A Long-Term Study Evaluating the Effects of Nicorandil Treatment on Duchenne Muscular Dystrophy-Associated Cardiomyopathy in mdx Mice.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by dystrophin gene mutations affecting striated muscle. Due to advances in skeletal muscle treatment, cardiomyopathy has emerged as a leading cause of death. Previously, nicorandil, a drug with antioxidant and nitrate-like properties, ameliorated cardiac damage and improved cardiac function in young, injured mdx mice. Nicorandil mitigated damage by stimulating antioxidant activity and limiting pro-oxidant expression. Here, we examined whether nicorandil was similarly cardioprotective in aged mdx mice. METHODS AND RESULTS: Nicorandil (6 mg/kg) was given over 15 months. Echocardiography of mdx mice showed some functional defects at 12 months compared to wild-type (WT) mice, but not at 15 months. Disease manifestation was evident in mdx mice via treadmill assays and survival, but not open field and grip strength assays. Cardiac levels of SOD2 and NOX4 were decreased in mdx vs. WT. Nicorandil increased survival in mdx but did not alter cardiac function, fibrosis, diaphragm function or muscle fatigue. CONCLUSIONS: In contrast to our prior work in young, injured mdx mice, nicorandil did not exert cardioprotective effects in 15 month aged mdx mice. Discordant findings may be explained by the lack of cardiac disease manifestation in aged mdx mice compared to WT, whereas significant cardiac dysfunction was previously seen with the sub-acute injury in young mice. Therefore, we are not able to conclude any cardioprotective effects with long-term nicorandil treatment in aging mdx mice.

Optimizing the Differentiation of Cardiomyocytes from Human Induced Pluripotent-Derived Stem Cells.

Cardiovascular disease is a worldwide health issue that affects millions of lives every year, and thus, researchers are in need of high-throughput model systems with which to investigate mechanisms of disease and to develop and test potential therapies. The use of human-derived induced pluripotent stem cells (iPSCs) differentiated into cardiomyocytes aims to address this need. While cardiac differentiation protocols have been established previously in iPSCs, optimization of cardiac differentiation remains crucial to obtaining high quality cardiomyocytes for future experimental analyses. Important factors to consider include cell density and rate of proliferation, temporal regulation of media changes throughout the differentiation process, and the concentration of the chemicals utilized. In this chapter, we present a detailed protocol to outline the process of differentiating cardiomyocytes from human iPSCs via modulation of Wnt signaling, characterization of cardiomyocytes by immunofluorescence, as well as guidelines for troubleshooting and optimizing these techniques.

Identification of Cardiac Fibrosis in Young Adults With a Homozygous Frameshift Variant in SERPINE1.

Importance: Cardiac fibrosis is exceedingly rare in young adults. Identification of genetic variants that cause early-onset cardiomyopathy may inform novel biological pathways. Experimental models and a single case report have linked genetic deficiency of plasminogen activator inhibitor-1 (PAI-1), a downstream target of cardiac transforming growth factor ß, with cardiac fibrosis. Objective: To perform detailed cardiovascular phenotyping and genotyping in young adults from an Amish family with a frameshift variant (c.699_700dupTA) in SERPINE1, the gene that codes for PAI-1. Design, Setting, and Participants: This observational study included participants from 3 related nuclear families from an Amish community in the primary analysis and participants from the extended family in the secondary analysis. Participants were recruited from May 2015 to December 2016, and analysis took place from June 2015 to June 2020. Main Outcomes and Measures: (1) Multimodality cardiovascular imaging (transthoracic echocardiography and cardiac magnetic resonance imaging), (2) whole-exome sequencing, and (3) induced pluripotent stem cell-derived cardiomyocytes. Results: Among 17 participants included in the primary analysis, the mean (interquartile range) age was 23.7 (20.9-29.9) years and 9 individuals (52.9%) were confirmed to be homozygous for the SERPINE1 c.699_700dupTA variant. Late gadolinium enhancement was present in 6 of 9 homozygous participants (67%) with absolute PAI-1 deficiency vs 0 of 8 in the control group (P = .001). Late gadolinium enhancement patterns tended to be dense and linear, usually subepicardial but also midmyocardial and transmural with noncoronary distributions. Targeted whole-exome sequencing analysis identified that homozygosity for c.699_700dupTA SERPINE1 was the only shared pathogenic variant or variant of uncertain significance after examination of cardiomyopathy genes among those with late gadolinium enhancement. Induced pluripotent stem cell-derived cardiomyocytes from participants homozygous for the SERPINE1 c.699_700dupTA variant exhibited susceptibility to cardiomyocyte injury in response to angiotensin II (increased transforming growth factor ß1 secretion and release of lactate dehydrogenase) compared with control induced pluripotent stem cell-derived cardiomyocytes. In a secondary analysis based on echocardiography in 155 individuals across 3 generations in the extended family, no difference in global longitudinal strain was observed in carriers for the SERPINE1 c.699_700dupTA variant compared with wild-type participants, supporting an autosomal recessive inheritance pattern. Conclusions and Relevance: In this study, a highly penetrant, autosomal recessive, cardiac fibrosis phenotype among young adults with homozygous frameshift variant for SERPINE1 was identified, suggesting an optimal range of PAI-1 levels are needed for cardiac homeostasis.

Parstatin(1-26): the putative signal peptide of protease-activated receptor 1 confers potent protection from myocardial ischemia-reperfusion injury.

Parstatin, the N-terminal 41-amino-acid peptide cleaved by thrombin from the protease-activated receptor 1, protects against rat myocardial ischemia and reperfusion injury. In this study, we determined that the parstatin fragment 1-26, the putative signal peptide of protease-activated receptor 1, contains the functional domain of parstatin. We assessed a synthesized parstatin(1-26) peptide in an in vivo rat model of myocardial regional ischemia-reperfusion injury (n = 6/group). Infarct size in control rat hearts was 58 +/- 1% area at risk. Parstatin(1-26) was able to reduce infarct size to 13 +/- 1% (P < 0.001) and 22 +/- 1% area at risk (P < 0.01) when given before or after reperfusion. The infarct-sparing effects of parstatin(1-26) were abolished by inhibition of G(i) proteins (pertussis toxin), phosphoinositide 3-kinase/Akt (wortmannin), nitric-oxide synthase (NOS; N(G)-monomethyl-l-arginine), soluble guanylyl cyclase [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)], and sarcolemmal and mitochondrial K(ATP) channels [glibenclamide, 5-hydroxydecanoic acid, and sodium (5-(2-(5-chloro-2-methoxybenzamido)ethyl)-2-methoxyphenylsulfonyl) (methylcarbamothioyl)amide (HMR 1098)]. Parstatin(1-26) cardioprotection was also abolished by atractyloside, a mitochondrial permeability transition pore (mPTP) opener. The inhibitors and opener alone had no effect on infarct size. Furthermore, preischemic treatment with parstatin(1-26) increased Akt and endothelial NOS phosphorylation at the time of reperfusion. After a 120-min reperfusion, parstatin(1-26) increased nitric oxide levels (12 +/- 0.4 to 17 +/- 0.9 mmol/g tissue) and cyclic GMP levels (87 +/- 21 to 395 +/- 36 pmol/g tissue). Parstatin(1-26) treatment either before or after ischemia results in an extremely efficacious protection against ischemia-reperfusion injury that depends on a G(i) protein-mediated pathway involving mPTP, the end effector of the preconditioning pathway. This suggests that parstatin(1-26) has a potential therapeutic role in the treatment of ischemia and reperfusion injury.

Duchenne muscular dystrophy (DMD) cardiomyocyte-secreted exosomes promote the pathogenesis of DMD-associated cardiomyopathy.

Cardiomyopathy is a leading cause of early mortality in Duchenne muscular dystrophy (DMD). There is a need to gain a better understanding of the molecular pathogenesis for the development effective therapies. Exosomes (exo) are secreted vesicles and exert effects via their RNA, lipid and protein cargo. The role of exosomes in disease pathology is unknown. Exosomes derived from stem cells have demonstrated cardioprotection in the murine DMD heart. However, it is unknown how the disease status of the donor cell type influences exosome function. Here, we sought to determine the phenotypic responses of DMD cardiomyocytes (DMD-iCMs) after long-term exposure to DMD cardiac exosomes (DMD-exo). DMD-iCMs were vulnerable to stress, evidenced by production of reactive oxygen species, the mitochondrial membrane potential and cell death levels. Long-term exposure to non-affected exosomes (N-exo) was protective. By contrast, long-term exposure to DMD-exo was not protective, and the response to stress improved with inhibition of DMD-exo secretion in vitro and in vivo The microRNA (miR) cargo, but not exosome surface peptides, was implicated in the pathological effects of DMD-exo. Exosomal surface profiling revealed N-exo peptides associated with PI3K-Akt signaling. Transcriptomic profiling identified unique changes with exposure to either N- or DMD-exo. Furthermore, DMD-exo miR cargo regulated injurious pathways, including p53 and TGF-beta. The findings reveal changes in exosomal cargo between healthy and diseased states, resulting in adverse outcomes. Here, DMD-exo contained miR changes, which promoted the vulnerability of DMD-iCMs to stress. Identification of these molecular changes in exosome cargo and effectual phenotypes might shed new light on processes underlying DMD cardiomyopathy.This article has an associated First Person interview with the first author of the paper.

A Pilot Study of Cardiac MRI in Breast Cancer Survivors After Cardiotoxic Chemotherapy and Three-Dimensional Conformal Radiotherapy.

PURPOSE/OBJECTIVES: Node-positive breast cancer patients often receive chemotherapy and regional nodal irradiation. The cardiotoxic effects of these treatments, however, may offset some of the survival benefit. Cardiac magnetic resonance (CMR) is an emerging modality to assess cardiac injury. This is a pilot trial assessing cardiac damage using CMR in patients who received anthracycline-based chemotherapy and three-dimensional conformal radiotherapy (3DCRT) regional nodal irradiation using heart constraints. MATERIALS AND METHODS: Node-positive breast cancer patients (2000-2008) treated with anthracycline-based chemotherapy and 3DCRT regional nodal irradiation (including the internal mammary chain nodes) with heart ventricular constraints (V25 < 10%) were invited to participate. Cardiac tissues were contoured and analyzed separately for whole heart (pericardium) and for combined ventricles and left atrium (myocardium). CMR obtained ventricular function/dimensions, late gadolinium enhancement (LGE), global longitudinal strain (GLS), and extracellular volume fraction (ECV) as measures of cardiac injury and/or early fibrosis. CMR parameters were correlated with dose-volume constraints using Spearman correlations. RESULTS: Fifteen left-sided and five right-sided patients underwent CMR. Median diagnosis age was 50 (32-77). No patients had baseline cardiac disease before regional nodal irradiation. Median time after 3DCRT was 8.3 years (5.2-14.4). Median left-sided mean heart dose (MHD) was 4.8 Gy (1.1-11.2) and V25 was 5.7% (0-12%). Median left ventricular ejection fraction (LVEF) was 63%. No abnormal LGE was observed. No correlations were seen between whole heart doses and LVEF, LV mass, GLS, or LV dimensions. Increasing ECV did not correlate with increased heart or ventricular doses. However, correlations between higher LV mass and ventricular mean dose, V10, and V25 were seen. CONCLUSION: At a median follow-up of 8.3 years, this cohort of node-positive breast cancer patients who received anthracycline-based chemotherapy and regional nodal irradiation had no clinically abnormal CMR findings. However, correlations between ventricular mean dose, V10, and V25 and LV mass were seen. Larger corroborating studies that include advanced techniques for measuring regional heart mechanics are warranted.

Human thrombopoietin reduces myocardial infarct size, apoptosis, and stunning following ischaemia/reperfusion in rats.

AIMS: Thrombopoietin (Tpo) is known for its ability to stimulate platelet production. However, it is currently unknown whether Tpo plays a physiological function in the heart. METHODS AND RESULTS: We assessed the potential protective role of Tpo in vitro and in vivo in two rat models of myocardial ischaemia/reperfusion. Tpo receptor (c-mpl) message was detected in the heart using RT-PCR, and the Tpo receptor protein was detected using western blotting and immunohistochemistry. Tpo treatment immediately before ischaemia reduced myocardial necrosis, apoptosis, and decline in ventricular function following ischaemia/reperfusion in the rat in a concentration- and dose-dependent manner with an optimal concentration of 1.0 ng/mL in vitro and an optimal dose of 0.05 microg/kg iv in vivo. Tpo also reduced infarct size when given after the onset of ischaemia or at reperfusion. Tpo activated JAK-2 (Janus kinase-2) and p44 MAPK (mitogen-activated protein kinase) during reperfusion but not prior to ischaemia. Inhibition of JAK-2 (AG-490), p42/44 MAPK (PD98059), mitochondrial K(ATP) channels (5-HD), and sarcolemmal K(ATP) channels (HMR 1098) abolished Tpo-induced resistance to injury from myocardial ischaemia/reperfusion. AG-490, PD98059, 5-HD, and HMR1098 alone had no effect on cardioprotection. Treatment with a single dose of Tpo (0.05 or 1.0 microg/kg iv) did not result in the elevation of platelet count or haematocrit over a 16-day period. CONCLUSION: A single treatment of Tpo confers cardioprotection through JAK-2, p42/44 MAPK, and K(ATP) channels, suggesting a potential therapeutic role of Tpo in the treatment of injury resulting from myocardial ischaemia and reperfusion.

Probenecid: An Oral Inotrope for End-Stage Heart Failure in a Case With Myotonic Dystrophy.

A 56-year-old man with multiple cardiac manifestations of type 1 myotonic dystrophy, including severe, nonischemic cardiomyopathy, presented in refractory cardiogenic shock requiring inotropic therapy. Given his wishes to die without having any intravenous medications, he was started on oral probenecid therapy, which allowed for successful elimination of his intravenous therapies. (Level of Difficulty: Intermediate.).