Outcomes of hypothalamic oxytocin neuron-driven cardioprotection after acute myocardial infarction.

Altered autonomic balance is a hallmark of numerous cardiovascular diseases, including myocardial infarction (MI). Although device-based vagal stimulation is cardioprotective during chronic disease, a non-invasive approach to selectively stimulate the cardiac parasympathetic system immediately after an infarction does not exist and is desperately needed. Cardiac vagal neurons (CVNs) in the brainstem receive powerful excitation from a population of neurons in the paraventricular nucleus (PVN) of the hypothalamus that co-release oxytocin (OXT) and glutamate to excite CVNs. We tested if chemogenetic activation of PVN-OXT neurons following MI would be cardioprotective. The PVN of neonatal rats was transfected with vectors to selectively express DREADDs within OXT neurons. At 6 weeks of age, an MI was induced and DREADDs were activated with clozapine-N-oxide. Seven days following MI, patch-clamp electrophysiology confirmed the augmented excitatory neurotransmission from PVN-OXT neurons to downstream nuclei critical for parasympathetic activity with treatment (43.7 ± 10 vs 86.9 ± 9 pA; MI vs. treatment), resulting in stark improvements in survival (85% vs. 95%; MI vs. treatment), inflammation, fibrosis assessed by trichrome blue staining, mitochondrial function assessed by Seahorse assays, and reduced incidence of arrhythmias (50% vs. 10% cumulative incidence of ventricular fibrillation; MI vs. treatment). Myocardial transcriptomic analysis provided molecular insight into potential cardioprotective mechanisms, which revealed the preservation of beneficial signaling pathways, including muscarinic receptor activation, in treated animals. These comprehensive results demonstrate that the PVN-OXT network could be a promising therapeutic target to quickly activate beneficial parasympathetic-mediated cellular pathways within the heart during the early stages of infarction.

Hypothalamic Oxytocin Neuron Activation Attenuates Intermittent Hypoxia-Induced Hypertension and Cardiac Dysfunction in an Animal Model of Sleep Apnea.

BACKGROUND: Obstructive sleep apnea is a prevalent and poorly treated cardiovascular disease that leads to hypertension and autonomic imbalance. Recent studies that restore cardiac parasympathetic tone using selective activation of hypothalamic oxytocin neurons have shown beneficial cardiovascular outcomes in animal models of cardiovascular disease. This study aimed to determine if chemogenetic activation of hypothalamic oxytocin neurons in animals with existing obstructive sleep apnea-induced hypertension would reverse or blunt the progression of autonomic and cardiovascular dysfunction. METHODS: Two groups of rats were exposed to chronic intermittent hypoxia (CIH), a model of obstructive sleep apnea, for 4 weeks to induce hypertension. During an additional 4 weeks of exposure to CIH, 1 group was treated with selective activation of hypothalamic oxytocin neurons while the other group was untreated. RESULTS: Hypertensive animals exposed to CIH and treated with daily hypothalamic oxytocin neuron activation had lower blood pressure, faster heart rate recovery times after exercise, and improved indices of cardiac function compared with untreated hypertensive animals. Microarray analysis suggested that, compared with treated animals, untreated animals had gene expression profiles associated with cellular stress response activation, hypoxia-inducible factor stabilization, and myocardial extracellular matrix remodeling and fibrosis. CONCLUSIONS: In animals already presenting with CIH-induced hypertension, chronic activation of hypothalamic oxytocin neurons blunted the progression of hypertension and conferred cardioprotection after an additional 4 weeks of CIH exposure. These results have significant clinical translation for the treatment of cardiovascular disease in patients with obstructive sleep apnea.

Epigenetic Alterations in Pediatric Sleep Apnea.

Pediatric obstructive sleep apnea has significant negative effects on health and behavior in childhood including depression, failure to thrive, neurocognitive impairment, and behavioral issues. It is strongly associated with an increased risk for chronic adult disease such as obesity and diabetes, accelerated atherosclerosis, and endothelial dysfunction. Accumulating evidence suggests that adult-onset non-communicable diseases may originate from early life through a process by which an insult applied at a critical developmental window causes long-term effects on the structure or function of an organism. In recent years, there has been increased interest in the role of epigenetic mechanisms in the pathogenesis of adult disease susceptibility. Epigenetic mechanisms that influence adaptive variability include histone modifications, non-coding RNAs, and DNA methylation. This review will highlight what is currently known about the phenotypic associations of epigenetic modifications in pediatric obstructive sleep apnea and will emphasize the importance of epigenetic changes as both modulators of chronic disease and potential therapeutic targets.

Activation of Oxytocin Neurons Improves Cardiac Function in a Pressure-Overload Model of Heart Failure.

This work shows long-term restoration of the hypothalamic oxytocin (OXT) network preserves OXT release, reduces mortality, cardiac inflammation, fibrosis, and improves autonomic tone and cardiac function in a model of heart failure. Intranasal administration of OXT in patients mimics the short-term changes seen in animals by increasing parasympathetic-and decreasing sympathetic-cardiac activity. This work provides the essential translational foundation to determine if approaches that mimic paraventricular nucleus (PVN) OXT neuron activation, such as safe, noninvasive, and well-tolerated intranasal administration of OXT, can be beneficial in patients with heart failure.

Protein kinase C binding protein 1 inhibits hypoxia-inducible factor-1 in the heart.

AIMS: Hypoxia-inducible factor-1 alpha (HIF-1α) is a key transcription factor responsible for the induction of genes that facilitate adaptation to hypoxia. To study HIF-1 signalling in the heart, we developed a mouse model in which an oxygen-stable form of HIF-1α can be inducibly expressed in cardiac myocytes, under the regulation of tetracycline. METHODS AND RESULTS: Remarkably, expression of the transgene in mice generated two distinct phenotypes. One was the expected expression of HIF-regulated transcripts and associated changes in cardiac angiogenesis and contractility. The other was an unresponsive phenotype with much less expression of typical HIF-response genes and substantial expression of a zinc-finger protein, Protein Kinase C Binding Protein 1 (PRKCBP1). We have demonstrated that this second phenotype is due to an insertion of a fragment of DNA upstream of the PRKCBP1 gene that contains two additional canonical HIF binding sites and leads to substantial HIF binding, assessed by chromatin immunoprecipitation, and transcriptional activation. This insertion is found only in the FVB strain of mice that contributed the αMHC-tet binding protein transgene to these biallelic mice. In HEK293 cells transfected with oxygen-stable HIF-1α and PRKCBP1, we demonstrated inhibition of HIF-1 activity by a luciferase reporter assay. Using mouse primary cells and cell lines, we show that transfection with oxygen-stable HIF-1α and PRKCBP1 reduced expression of direct HIF-1 gene targets and that knockdown of PRKCBP1 removes that negative inhibition. Consistent with previous reports suggesting that PRKCBP1 modulates the chromatin landscape, we found that HL-1 cells transfected with oxygen-stable HIF-1α and PRKCBP1 have reduced global 5-methyl cytosine compared to HIF-1 alone. CONCLUSION: We show genetic, transcriptional, biochemical, and physiological evidence that PRKCBP1 inhibits HIF activity. Identification of a new oxygen-dependent and previously unsuspected regulator of HIF may provide a target for new therapeutic approaches to ischaemic heart disease.

HIF1 mediates a switch in pyruvate kinase isoforms after myocardial infarction.

Alternative splicing of RNA is an underexplored area of transcriptional response. We expect that early changes in alternatively spliced genes may be important for responses to cardiac injury. Hypoxia inducible factor 1 (HIF1) is a key transcription factor that rapidly responds to loss of oxygen through alteration of metabolism and angiogenesis. The goal of this study was to investigate the transcriptional response after myocardial infarction (MI) and to identify novel, hypoxia-driven changes, including alternative splicing. After ligation of the left anterior descending artery in mice, we observed an abrupt loss of cardiac contractility and upregulation of hypoxic signaling. We then performed RNA sequencing on ischemic heart tissue 1 and 3 days after infarct to assess early transcriptional changes and identified 89 transcripts with altered splicing. Of particular interest was the switch in Pkm isoform expression (pyruvate kinase, muscle). The usually predominant Pkm1 isoform was less abundant in ischemic hearts, while Pkm2 and associated splicing factors (hnRNPA1, hnRNPA2B1, Ptbp1) rapidly increased. Despite increased Pkm2 expression, total pyruvate kinase activity remained reduced in ischemic myocardial tissue. We also demonstrated HIF1 binding to PKM by chromatin immunoprecipitation, indicating a direct role for HIF1 in mediating this isoform switch. Our study provides a new, detailed characterization of the early transcriptome after MI. From this analysis, we identified an HIF1-mediated alternative splicing event in the PKM gene. Pkm1 and Pkm2 play distinct roles in glycolytic metabolism and the upregulation of Pkm2 is likely to have important consequences for ATP synthesis in infarcted cardiac muscle.

Long-term analysis of 2 prospective studies that incorporate mitomycin C into an adjuvant chemoradiation regimen for pancreatic and periampullary cancers.

PURPOSE: The purpose of this study was to report toxicity and long-term survival outcomes of 2 prospective trials evaluating mitomycin C (MMC) with 5-fluorouracil-based adjuvant chemoradiation in resected periampullary adenocarcinoma. METHODS AND MATERIALS: From 1996 to 2002, 119 patients received an adjuvant 4-drug chemotherapy regimen of 5-fluorouracil, leucovorin, MMC, and dipyridamole with chemoradiation on 2 consecutive trials (trials A and B). Trial A patients received upfront chemoradiation (50 Gy split-course, 2.5 Gy/fraction) followed by 4 cycles of the 4-drug chemotherapy with bolus 5-fluorouracil. Trial B patients received 1 cycle of the 4-drug chemotherapy with continuous infusion 5-fluorouracil followed by continuous chemoradiation (45-54 Gy, 1.8 Gy/fraction) and 2 additional cycles of chemotherapy. Cox proportional hazards models were performed to identify prognostic factors for overall survival (OS). RESULTS: Of the 62 trial A patients, 61% had pancreatic and 39% nonpancreatic periampullary carcinomas. Trial B (n = 57) consisted of 68% pancreatic and 32% nonpancreatic periampullary carcinomas. Resection margin and lymph node status were similar for both trials. Median follow-up was longer for trial A than trial B (197.5 vs 107.0 months), with median OS of 32.2 and 24.2 months, respectively. Rates of 3-, 5-, and 10-year OS were 48%, 31%, and 26% in trial A and 32%, 23%, and 9% in trial B. On multivariate analysis, lymph node-positive resection was the strongest prognostic factor for OS. A pancreatic primary and positive margin status were also associated with inferior survival (P < .05). Rates of grade ≥3 treatment-related toxicity in trials A and B were 2% and 7%, respectively. CONCLUSIONS: This is the first study to report long-term outcomes of MMC with 5-fluorouracil-based adjuvant chemoradiation in periampullary cancers. Because MMC may be considered in DNA repair-deficient carcinomas, randomized trials are needed to determine the true benefit of adjuvant MMC.

Separating fast and slow exchange transfer and magnetization transfer using off-resonance variable-delay multiple-pulse (VDMP) MRI.

PURPOSE: To develop a method that can separate and quantify the fast (>1 kHz) and slow exchange transfer and magnetization transfer components in Z-spectra. METHODS: Z-spectra were recorded as a function of mixing time using a train of selective pulses providing variable-delay multipulse build-up curves. Fast and slow transfer components in the Z-spectra were separated and quantified on a voxel-by-voxel basis by fitting the mixing time-dependent CEST signal using a 3-pool model. RESULTS: Phantom studies of glutamate solution, bovine serum albumin solution, and hair conditioner showed the capability of the proposed method to separate fast and slow transfer components. In vivo mouse brain studies showed a strong contrast between white matter and gray matter in the slow-transferring map, corresponding to an asymmetric component of the conventional semisolid magnetization transfer contrast. In addition, a fast-transferring proton map was found that was homogeneous across the brain and attributed to the total contributions of the fast-exchanging protons from proteins, metabolites, and a symmetric magnetization transfer contrast component. CONCLUSIONS: This new method provides a simple way to extract fast and slow transfer components from the Z-spectrum, leading to novel MRI contrasts, and providing insight into the different magnetization transfer contrast contributions.

A novel atherothrombotic model of ischemic stroke induced by injection of collagen into the cerebral vasculature.

BACKGROUND: Most ischemic strokes in humans are caused by ruptured arterial atheroma, which activate platelets and produce thrombi that occlude cerebral vessels. METHODS: To simulate these events, we threaded a catheter through the internal carotid artery toward the middle cerebral artery (MCA) orifice and injected collagen directly into the cerebral circulation of male C57Bl/6 mice and Wistar rats. RESULTS: Laser-Doppler flowmetry demonstrated reductions in cerebral blood flow (CBF) of ∼80% in mice and ∼60% in rats. CBF spontaneously increased but remained depressed after catheter withdrawal. Magnetic resonance imaging showed that ipsilateral CBF was reduced at 3h after collagen injection and markedly improved at 48 h. Micro-computed tomography revealed reduced blood vessel density in the ipsilateral MCA territory at 3 h. Gross examination of excised brains revealed thrombi within ipsilateral cerebral arteries at 3 h, but not 24 h, after collagen injection. Immunofluorescence microscopy confirmed that platelets and fibrinogen/fibrin were major components of these thrombi at both macrovascular and microvascular levels. Cerebral infarcts comprising ∼30% of hemispheric volume and neurobehavioral deficits were observed 48 h after ischemic injury in both mice and rats. COMPARISON WITH EXISTING METHODS: Collagen injection caused brain injury that was similar in magnitude and variability to mechanical MCA occlusion or injection of a pre-formed clot; however, alterations in CBF and the mechanism of vascular occlusion were more consistent with clinical ischemic stroke. CONCLUSION: This novel rodent model of ischemic stroke has pathophysiologic characteristics consistent with clinical atherothrombotic stroke, is technically feasible, and creates reproducible brain injury.

Cathepsin G-dependent modulation of platelet thrombus formation in vivo by blood neutrophils.

Neutrophils are consistently associated with arterial thrombotic morbidity in human clinical studies but the causal basis for this association is unclear. We tested the hypothesis that neutrophils modulate platelet activation and thrombus formation in vivo in a cathepsin G-dependent manner. Neutrophils enhanced aggregation of human platelets in vitro in dose-dependent fashion and this effect was diminished by pharmacologic inhibition of cathepsin G activity and knockdown of cathepsin G expression. Tail bleeding time in the mouse was prolonged by a cathepsin G inhibitor and in cathepsin G knockout mice, and formation of neutrophil-platelet conjugates in blood that was shed from transected tails was reduced in the absence of cathepsin G. Bleeding time was highly correlated with blood neutrophil count in wildtype but not cathepsin G deficient mice. In the presence of elevated blood neutrophil counts, the anti-thrombotic effect of cathepsin G inhibition was greater than that of aspirin and additive to it when administered in combination. Both pharmacologic inhibition of cathepsin G and its congenital absence prolonged the time for platelet thrombus to form in ferric chloride-injured mouse mesenteric arterioles. In a vaso-occlusive model of ischemic stroke, inhibition of cathepsin G and its congenital absence improved cerebral blood flow, reduced histologic brain injury, and improved neurobehavioral outcome. These experiments demonstrate that neutrophil cathepsin G is a physiologic modulator of platelet thrombus formation in vivo and has potential as a target for novel anti-thrombotic therapies.

Acetaminophen attenuates doxorubicin-induced cardiac fibrosis via osteopontin and GATA4 regulation: reduction of oxidant levels.

It is well documented in animal and human studies that therapy with the anti-cancer drug doxorubicin (DOX) induces fibrosis, cardiac dysfunction, and cell death. The most widely accepted mechanism of cardiac injury is through production of reactive oxygen species (ROS), which cause mitochondrial damage, sarcomere structural alterations, and altered gene expression in myocytes and fibroblasts. Here we investigated the effects of acetaminophen (APAP, N-acetyl-para-aminophenol) on DOX-induced cardiac injury and fibrosis in the presence or absence of osteopontin (OPN). H9c2 rat heart-derived embryonic myoblasts were exposed to increasing concentrations of DOX ± APAP; cell viability, oxidative stress, and OPN transcript levels were analyzed. We found a dose-dependent decrease in cell viability and a corresponding increase in intracellular oxidants at the tested concentrations of DOX. These effects were attenuated in the presence of APAP. RT-PCR analysis revealed a small increase in OPN transcript levels in response to DOX, which was suppressed by APAP. When male 10-12-week-old mice (OPN(+/+) or OPN(-/-)) were given weekly injections of DOX ± APAP for 4 weeks there was substantial cardiac fibrosis in OPN(+/+) and, to a lesser extent, in OPN(-/-) mice. In both groups, APAP decreased fibrosis to near baseline levels. Activity of the pro-survival GATA4 transcription factor was diminished by DOX in both mouse genotypes, but retained baseline activity in the presence of APAP. These effects were mediated, in part, by the ability of APAP, acting as an anti-inflammatory agent, to decrease intracellular ROS levels, consequently diminishing the injury-induced increase in OPN levels.