Cardiac and skeletal muscle manifestations in the G608G mouse model of Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare premature aging disorder resulting from de novo mutations in the lamin A gene. Children with HGPS typically pass away in their teenage years due to cardiovascular diseases such as atherosclerosis, myocardial infarction, heart failure, and stroke. In this study, we characterized the G608G HGPS mouse model and explored cardiac and skeletal muscle function, along with senescence-associated phenotypes in fibroblasts. Homozygous G608G HGPS mice exhibited cardiac dysfunction, including decreased cardiac output and stroke volume, and impaired left ventricle relaxation. Additionally, skeletal muscle exhibited decreased isometric tetanic torque, muscle atrophy, and increased fibrosis. HGPS fibroblasts showed nuclear abnormalities, decreased proliferation, and increased expression of senescence markers. These findings provide insights into the pathophysiology of the G608G HGPS mouse model and inform potential therapeutic strategies for HGPS.

Rejuvenating effects of young extracellular vesicles in aged rats and in cellular models of human senescence.

Rejuvenation of an old organism was achieved in heterochronic parabiosis experiments, implicating different soluble factors in this effect. Extracellular vesicles (EVs) are the secretory effectors of many cells, including cardiosphere-derived cells (CDCs) with demonstrated anti-senescent effect. 1. To determine the role of EVs (versus other blood fractions) on the rejuvenating effect of the young blood. 2. To evaluate the anti-aging properties of therapeutically administered EVs secreted by young-CDCs in an old organism. Neonatal blood fractioned in 4 components (whole blood, serum, EV-depleted serum and purified EVs) was used to treat old human cardiac stromal cells (CSPCs). CDCs were generated from neonatal rat hearts and the secreted CDC-EVs were purified. CDC-EVs were then tested in naturally-aged rats, using monthly injections over 4-months period. For validation in human samples, pediatric CDC-EVs were tested in aged human CSPCs and progeric fibroblasts. While the purified EVs reproduced the rejuvenating effects of the whole blood, CSPCs treated with EV-depleted serum exhibited the highest degree of senescence. Treatment with young CDC-EVs induce structural and functional improvements in the heart, lungs, skeletal muscle, and kidneys of old rats, while favorably modulating glucose metabolism and anti-senescence pathways. Lifespan was prolonged. EVs secreted by young CDCs exert broad-ranging anti-aging effects in aged rodents and in cellular models of human senescence. Our work not only identifies CDC-EVs as possible therapeutic candidates for a wide range of age-related pathologies, but also raises the question of whether EVs function as endogenous modulators of senescence.

Mass murders involving firearms and other methods in school, college, and university settings: Findings from the Columbia Mass Murder Database.

While mass murders involving academic settings, especially using firearms, are of grave, growing public concern, identifying consistent patterns to aid prevention has proved challenging. Although some characteristics, such as male sex, have been routinely associated with these events, another hypothesized risk factor, severe mental illness, has been less reliably predictive. We isolated cases of mass murder perpetrated at least in part at schools, colleges, and universities from the Columbia Mass Murder Database (CMDD) and categorized them by location (within or outside of the US), and whether firearms were used. Demographic similarities and differences between groups were analyzed statistically wherever possible. We examined 82 incidents of mass murder, by any means, involving academic settings. Nearly half of all incidents (47.6%), and most involving firearms (63.2%), were U.S.-based, whereas those not involving firearms largely occurred elsewhere (88.0%). Consistent with previous reports, perpetrators of mass shootings involving academic settings are primarily Caucasian (66.7%) and male (100%). Severe mental illness (i.e., psychosis) was absent in the majority of perpetrators (firearms: 80.7%; nonfirearms: 68.0%). About half (45.6%) of mass school shootings ended with the perpetrator's suicide. When present, psychotic symptoms are more associated with mass murders in academic settings involving means other than firearms. The question of whether perpetrators of such incidents may perceive their actions as a kind of final act might enhance policy development and/or how law enforcement intervenes.

MicroRNA-dependent suppression of biological pacemaker activity induced by TBX18.

Chemically modified mRNA (CMmRNA) with selectively altered nucleotides are used to deliver transgenes, but translation efficiency is variable. We have transfected CMmRNA encoding human T-box transcription factor 18 (CMmTBX18) into heart cells or the left ventricle of rats with atrioventricular block. TBX18 protein expression from CMmTBX18 is weak and transient, but Acriflavine, an Argonaute 2 inhibitor, boosts TBX18 levels. Small RNA sequencing identified two upregulated microRNAs (miRs) in CMmTBX18-transfected cells. Co-administration of miR-1-3p and miR-1b antagomiRs with CMmTBX18 prolongs TBX18 expression in vitro and in vivo and is sufficient to generate electrical stimuli capable of pacing the heart. Different suppressive miRs likewise limit the expression of VEGF-A CMmRNA. Cells therefore resist translation of CMmRNA therapeutic transgenes by upregulating suppressive miRs. Blockade of suppressive miRs enhances CMmRNA expression of genes driving biological pacing or angiogenesis. Such counterstrategies constitute an approach to boost the efficacy and efficiency of CMmRNA therapies.

Biological substrate modification suppresses ventricular arrhythmias in a porcine model of chronic ischaemic cardiomyopathy.

AIMS: Cardiomyopathy patients are prone to ventricular arrhythmias (VA) and sudden cardiac death. Current therapies to prevent VA include radiofrequency ablation to destroy slowly conducting pathways of viable myocardium which support re-entry. Here, we tested the reverse concept, namely that boosting local tissue viability in zones of slow conduction might eliminate slow conduction and suppress VA in ischaemic cardiomyopathy. METHODS AND RESULTS: Exosomes are extracellular vesicles laden with bioactive cargo. Exosomes secreted by cardiosphere-derived cells (CDCEXO) reduce scar and improve heart function after intramyocardial delivery. In a VA-prone porcine model of ischaemic cardiomyopathy, we injected CDCEXO or vehicle into zones of delayed conduction defined by electroanatomic mapping. Up to 1-month post-injection, CDCEXO, but not the vehicle, decreased myocardial scar, suppressed slowly conducting electrical pathways, and inhibited VA induction by programmed electrical stimulation. In silico reconstruction of electrical activity based on magnetic resonance images accurately reproduced the suppression of VA inducibility by CDCEXO. Strong anti-fibrotic effects of CDCEXO, evident histologically and by proteomic analysis from pig hearts, were confirmed in a co-culture assay of cardiomyocytes and fibroblasts. CONCLUSION: Biological substrate modification by exosome injection may be worth developing as a non-destructive alternative to conventional ablation for the prevention of recurrent ventricular tachyarrhythmias.

Extracellular vesicles from immortalized cardiosphere-derived cells attenuate arrhythmogenic cardiomyopathy in desmoglein-2 mutant mice.

AIMS: Arrhythmogenic cardiomyopathy (ACM) is characterized by progressive loss of cardiomyocytes, and fibrofatty tissue replacement. Extracellular vesicles (EVs) secreted by cardiosphere-derived cells, immortalized, and engineered to express high levels of ß-catenin, exert anti-inflammatory, and anti-fibrotic effects. The aim of the current study was to assess efficacy of EVs in an ACM murine model. METHODS AND RESULTS: Four-week-old homozygous knock-in mutant desmoglein-2 (Dsg2mt/mt) were randomized to receive weekly EVs or vehicle for 4 weeks. After 4 weeks, DSG2mt/mt mice receiving EVs showed improved biventricular function (left, P < 0.0001; right, P = 0.0037) and less left ventricular dilation (P < 0.0179). Electrocardiography revealed abbreviated QRS duration (P = 0.0003) and QTc interval (P = 0.0006) in EV-treated DSG2mt/mt mice. Further electrophysiology testing in the EV group showed decreased burden (P = 0.0042) and inducibility of ventricular arrhythmias (P = 0.0037). Optical mapping demonstrated accelerated repolarization (P = 0.0290) and faster conduction (P = 0.0274) in Dsg2mt/mt mice receiving EVs. DSG2mt/mt hearts exhibited reduced fibrosis, less cell death, and preserved connexin 43 expression after EV treatment. Hearts of Dsg2mt/mt mice expressed markedly increased levels of inflammatory cytokines that were, in part, attenuated by EV therapy. The pan-inflammatory transcription factor nuclear factor-κB (NF-κB), the inflammasome sensor NLRP3, and the macrophage marker CD68 were all reduced in EV-treated animals. Blocking EV hsa-miR-4488 in vitro and in vivo reactivates NF-κB and blunts the beneficial effects of EVs. CONCLUSIONS: Extracellular vesicle treatment improved cardiac function, reduced cardiac inflammation, and suppressed arrhythmogenesis in ACM. Further studies are needed prior to translating the present findings to human forms of this heterogenous disease.

Regulatory T cell activation, proliferation, and reprogramming induced by extracellular vesicles.

BACKGROUND: Extracellular vesicles (EVs) from heart stromal/progenitor cells modulate innate immunity, with salutary effects in a variety of cardiac disease models. Little is known, however, about the effects of these EVs on adaptive immunity. METHODS: Ex vivo differentiation of naïve CD4+ T cells was conducted to assess the effect of EVs on cytokine production and proliferation of Th1, Th2, Th17, and regulatory T (Treg) cells. These effects were further tested in vivo using the experimental autoimmune myocarditis (EAM) model. RESULTS: Using differentiated CD4+ T cells, we show that EVs secreted by human-derived heart stromal/progenitor cells selectively influence the phenotype, activity, and proliferation of regulatory T (Treg) cells. Exposure of Treg cells to EVs results in faster proliferation, augmented production of IL-10, and polarization toward an intermediate FOXP3+RORγt+ phenotype. In experimental autoimmune myocarditis, EVs attenuate cardiac inflammation and functional decline, in association with increased numbers of splenic IL10+-Treg cells. CONCLUSIONS: T cell modulation by EVs represents a novel therapeutic approach to inflammation, harnessing endogenous immunosuppressive mechanisms that may be applied in solid organ transplantation, graft-versus-host disease, and autoimmune disorders.

Cardiosphere-derived cells, with and without a biological scaffold, stimulate myogenesis and recovery of muscle function in mice with volumetric muscle loss.

Extremity trauma to military personnel and civilians commonly results in volumetric muscle loss (VML), leaving patients suffering chronic physical disability. Biomaterial-based technologies such as extracellular matrices (ECMs) are currently in clinical testing for soft tissue repair, but, in preclinical models of VML, the efficacy of ECMs is equivocal. In a murine model of VML, we investigated the effects of ECM and/or cardiosphere-derived cell (CDC) therapy; the latter improves skeletal myogenesis and muscle function in mdx mice, so we reasoned that CDCs may exert disease-modifying bioactivity in VML. While ECM alone improves functional recovery, CDCs have no additive or synergistic benefits with ECM transplantation following VML injury. However, CDCs alone are sufficient to promote muscle recovery, leading to sustained increases in muscle function throughout the study period. Notably, CDCs stimulate satellite cell accumulation in the muscle defect area and hasten myogenic progression (as evidenced by qPCR gene expression profiling), leading to global increases in myofiber numbers and anterior muscle compartment volume. Together, these data implicate CDCs as a viable therapeutic candidate to regenerate skeletal muscle injured by VML.

Extracellular Vesicles as Therapeutic Agents for Cardiac Fibrosis.

Heart disease remains an increasing major public health challenge in the United States and worldwide. A common end-organ feature in diseased hearts is myocardial fibrosis, which stiffens the heart and interferes with normal pump function, leading to pump failure. The development of cells for regenerative therapy has been met with many pitfalls on its path to clinical translation. Recognizing that regenerative cells secrete therapeutically bioactive vesicles has paved the way to circumvent many failures of cell therapy. In this review, we provide an overview of extracellular vesicles (EVs), with a focus on their utility as therapeutic agents for cardiac regeneration. We also highlight the engineering potential of EVs to enhance their therapeutic application.

Mechanisms of atrial fibrillation in aged rats with heart failure with preserved ejection fraction.

BACKGROUND: Although ∼20% of the elderly population develops atrial fibrillation (AF), little is known about the mechanisms. Heart failure with preserved ejection fraction (HFpEF), which is associated with AF, is more common in aged women than in men. OBJECTIVE: The purpose of this study was to identify potential mechanisms of AF in an age-related HFpEF model. METHODS: In aged female Fischer F344 rats (21- to 24-month-old), which are prone to HFpEF, we induced AF by atrial pacing. Young Fischer F344 female rats (3- to 4-month-old) and age-matched Sprague Dawley female rats (27-month-old) served as controls. Phenotyping included echocardiography to assess left ventricular structure/function; in vivo electrophysiology and ex vivo high-resolution optical mapping to assess AF vulnerability; systemic and atrial inflammatory profiling; atrial histology; and expression of inflammasome signaling proteins. RESULTS: Aged rats developed left ventricular hypertrophy, left atrial enlargement, diastolic dysfunction, and pulmonary congestion, without ejection fraction impairment, thus meeting the criteria for HFpEF. Increased serum inflammatory markers, hypertension, and obesity further characterize aged females. Sinoatrial and atrioventricular node dysfunction was associated with the high inducibility of AF in aged rats. Ex vivo electrical activation mapping revealed abnormal ß-adrenergic responsiveness and slowed conduction velocity. Atrial inflammasome signaling was enhanced in aged rats, which may contribute to fibrotic remodeling and high AF susceptibility. CONCLUSION: Together, our data demonstrate that aging-related atrial remodeling and HFpEF are associated with atrial enlargement, fibrosis, conduction abnormalities, and nodal dysfunction, favoring a substrate conducive to AF.

Disease-modifying bioactivity of intravenous cardiosphere-derived cells and exosomes in mdx mice.

Dystrophin deficiency leads to progressive muscle degeneration in Duchenne muscular dystrophy (DMD) patients. No known cure exists, and standard care relies on the use of antiinflammatory steroids, which are associated with side effects that complicate long-term use. Here, we report that a single intravenous dose of clinical-stage cardiac stromal cells, called cardiosphere-derived cells (CDCs), improves the dystrophic phenotype in mdx mice. CDCs augment cardiac and skeletal muscle function, partially reverse established heart damage, and boost the regenerative capacity of skeletal muscle. We further demonstrate that CDCs work by secreting exosomes, which normalize gene expression at the transcriptome level, and alter cell signaling and biological processes in mdx hearts and skeletal muscle. The work reported here motivated the ongoing HOPE-2 clinical trial of systemic CDC delivery to DMD patients, and identifies exosomes as next-generation cell-free therapeutic candidates for DMD.

Resistance exercise mediates remote ischemic preconditioning by limiting cardiac eNOS uncoupling.

BACKGROUND: Currently viewed as a complementary non-pharmacological intervention for preventing cardiac disorders, long-term aerobic training produces cardioprotection through remote ischemic preconditioning (RIPC) mechanisms. However, RIPC triggered by acute exercise remains poorly understood. Although resistance exercise (RE) has been highly recommended by several public health guidelines, there is no evidence showing that RE mediates RIPC. Hence, we investigated whether RE induces cardiac RIPC through nitric oxide synthase (NOS)-dependent mechanism. METHODS AND RESULTS: Acute RE at 40% of the maximal load augmented systemic nitrite levels, associated with increased cardiac eNOS phosphorylation, without affecting nNOS activity. Using an experimental model of myocardial infarction (MI) through ischemia-reperfusion (IR), RE fully prevented the loss of cardiac contractility and the extent of MI size compared to non-exercised (NE) rats. Moreover, RE mitigated aberrant ST-segment and reduced life-threatening arrhythmias induced by IR. Importantly, inhibition of NOS abolished the RE-mediated cardioprotection. After IR, NE rats showed increased cardiac eNOS activity, associated with reduced dimer/monomer ratio. Supporting the pivotal role of eNOS coupling during MI, non-exercised rats displayed a marked generation of reactive oxygen species (ROS) and oxidative-induced carbonylation of proteins, whereas RE prevented these responses. We validated our data demonstrating a restoration of physiological ROS levels in NE + IR cardiac sections treated with BH4, a cofactor oxidatively depleted during eNOS uncoupling, while cardiac ROS generation from exercised rats remained unchanged, suggesting no physiological needs of supplemental eNOS cofactors. CONCLUSION: Together, our findings strongly indicate that RE mediates RIPC by limiting eNOS uncoupling and mitigates myocardial IR injury.

Reverse electrical remodeling in rats with heart failure and preserved ejection fraction.

Sudden death is the most common mode of exodus in patients with heart failure and preserved ejection fraction (HFpEF). Cardiosphere-derived cells (CDCs) reduce inflammation and fibrosis in a rat model of HFpEF, improving diastolic function and prolonging survival. We tested the hypothesis that CDCs decrease ventricular arrhythmias (VAs) and thereby possibly contribute to prolonged survival. Dahl salt-sensitive rats were fed a high-salt diet to induce HFpEF. Allogeneic rat CDCs (or phosphate-buffered saline as placebo) were injected in rats with echo-verified HFpEF. CDC-injected HFpEF rats were less prone to VA induction by programmed electrical stimulation. Action potential duration (APD) was shortened, and APD homogeneity was increased by CDC injection. Transient outward potassium current density was upregulated in cardiomyocytes from CDC rats relative to placebo, as were the underlying transcript (Kcnd3) and protein (Kv4.3) levels. Fibrosis was attenuated in CDC-treated hearts, and survival was increased. Sudden death risk also trended down, albeit nonsignificantly. CDC therapy decreased VA in HFpEF rats by shortening APD, improving APD homogeneity, and decreasing fibrosis. Unlike other stem/progenitor cells, which often exacerbate arrhythmias, CDCs reverse electrical remodeling and suppress arrhythmogenesis in HFpEF.

Newt cells secrete extracellular vesicles with therapeutic bioactivity in mammalian cardiomyocytes.

Newts can regenerate amputated limbs and cardiac tissue, unlike mammals which lack broad regenerative capacity. Several signaling pathways involved in cell proliferation, differentiation and survival during newt tissue regeneration have been elucidated, however the factors that coordinate signaling between cells, as well as the conservation of these factors in other animals, are not well defined. Here we report that media conditioned by newt limb explant cells (A1 cells) protect mammalian cardiomyocytes from oxidative stress-induced apoptosis. The cytoprotective effect of A1-conditioned media was negated by exposing A1 cells to GW4869, which suppresses the generation of extracellular vesicles (EVs). A1-EVs are similar in diameter (~100-150 nm), structure, and share several membrane surface and cargo proteins with mammalian exosomes. However, isolated A1-EVs contain significantly higher levels of both RNA and protein per particle than mammalian EVs. Additionally, numerous cargo RNAs and proteins are unique to A1-EVs. Of particular note, A1-EVs contain numerous mRNAs encoding nuclear receptors, membrane ligands, as well as transcription factors. Mammalian cardiomyocytes treated with A1-EVs showed increased expression of genes in the PI3K/AKT pathway, a pivotal player in survival signaling. We conclude that newt cells secrete EVs with diverse, distinctive RNA and protein contents. Despite ~300 million years of evolutionary divergence between newts and mammals, newt EVs confer cytoprotective effects on mammalian cardiomyocytes.

Exosome-Mediated Benefits of Cell Therapy in Mouse and Human Models of Duchenne Muscular Dystrophy.

Genetic deficiency of dystrophin leads to disability and premature death in Duchenne muscular dystrophy (DMD), affecting the heart as well as skeletal muscle. Here, we report that clinical-stage cardiac progenitor cells, known as cardiosphere-derived cells (CDCs), improve cardiac and skeletal myopathy in the mdx mouse model of DMD. Injection of CDCs into the hearts of mdx mice augments cardiac function, ambulatory capacity, and survival. Exosomes secreted by human CDCs reproduce the benefits of CDCs in mdx mice and in human induced pluripotent stem cell-derived Duchenne cardiomyocytes. Surprisingly, CDCs and their exosomes also transiently restored partial expression of full-length dystrophin in mdx mice. The findings further motivate the testing of CDCs in Duchenne patients, while identifying exosomes as next-generation therapeutic candidates.

Resveratrol-Mediated Expression of KLF15 in the Ischemic Myocardium is Associated with an Improved Cardiac Phenotype.

PURPOSE: Myocardial infarction results in physiological derangements that lead to structural and functional alterations to the myocardium. In addition, oxidative stress potentiates cardiac remodeling and drives disease progression. Unfortunately, treatment with antioxidants in clinical trials have failed to show any therapeutic benefits despite the positive results reported in animal studies, which warrants further investigation into their mechanism(s) of action. Accordingly, the aim of this study was to elucidate a previously unknown mechanism of action for the antioxidant, resveratrol, in the treatment of the ischemic heart. METHODS: Male Sprague-Dawley rats underwent four weeks of chronic myocardial ischemia with or without daily resveratrol treatment (10 mg/kg/day). The expression and signaling of Krüppel-like factor 15 (KLF15) were determined by immunoblot and qPCR analyses, respectively. RESULTS: Chronic myocardial ischemia reduced the protein expression of KLF15. In parallel, mRNA transcripts of KLF15 gene targets actively involved in cardiac remodeling were robustly increased in untreated hearts. Importantly, daily treatment with resveratrol stimulated KLF15 expression, which was associated with attenuated gene expression and an improved cardiac phenotype. Additionally, we describe a novel role for KLF15 in the regulation of redox homeostasis. CONCLUSION: Based on our current findings, it appears that resveratrol treatment induces KLF15 expression, which may, in part, explain its therapeutic efficacy to improve the cardiac phenotype following ischemic injury.

Utility of 17-(allylamino)-17-demethoxygeldanamycin treatment for skeletal muscle injury.

Repeated eccentric contractions can injure skeletal muscle and result in functional deficits that take several weeks to fully recover. The 70-kDa heat shock protein (Hsp70) is a stress-inducible molecular chaperone that maintains protein quality and plays an integral role in the muscle's repair processes following injury. Here, we attempted to hasten this recovery by pharmacologically inducing Hsp70 expression in mouse skeletal muscle with 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) (40 mg/kg) both prior to and throughout the first 7 days after an injurious bout of 150 maximal eccentric contractions. Hsp70 content in the injured skeletal muscle was strongly induced following the eccentric contractions and remained elevated over the next 7 days as the muscle underwent repair. Treatment with 17-AAG increased Hsp70 content ∼fivefold; however, this was significantly less than that induced by the injury. Moreover, 17-AAG treatment did not recover the decrements to in vivo isometric torque production following the bout of eccentric contractions. Together, these findings demonstrate that although Hsp70 content was induced in the uninjured skeletal muscle, treatment of 17-AAG (40 mg/kg) was not a preventive measure to either reduce the severity of skeletal muscle damage or enhance functional recovery following a bout of maximal eccentric contractions.