RESUMO
Inherited cardiomyopathy caused by the p.(Arg14del) pathogenic variant of the phospholamban (PLN) gene is characterized by intracardiomyocyte PLN aggregation and can lead to severe dilated cardiomyopathy. We recently reported that pre-emptive depletion of PLN attenuated heart failure (HF) in several cardiomyopathy models. Here, we investigated if administration of a Pln-targeting antisense oligonucleotide (ASO) could halt or reverse disease progression in mice with advanced PLN-R14del cardiomyopathy. To this aim, homozygous PLN-R14del (PLN-R14 Δ/Δ) mice received PLN-ASO injections starting at 5 or 6 weeks of age, in the presence of moderate or severe HF, respectively. Mice were monitored for another 4 months with echocardiographic analyses at several timepoints, after which cardiac tissues were examined for pathological remodeling. We found that vehicle-treated PLN-R14 Δ/Δ mice continued to develop severe HF, and reached a humane endpoint at 8.1 ± 0.5 weeks of age. Both early and late PLN-ASO administration halted further cardiac remodeling and dysfunction shortly after treatment start, resulting in a life span extension to at least 22 weeks of age. Earlier treatment initiation halted disease development sooner, resulting in better heart function and less remodeling at the study endpoint. PLN-ASO treatment almost completely eliminated PLN aggregates, and normalized levels of autophagic proteins. In conclusion, these findings indicate that PLN-ASO therapy may have beneficial outcomes in PLN-R14del cardiomyopathy when administered after disease onset. Although existing tissue damage was not reversed, further cardiomyopathy progression was stopped, and PLN aggregates were resolved.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Cardiomiopatias/tratamento farmacológico , Oligonucleotídeos Antissenso/administração & dosagem , Substituição de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/química , Cardiomiopatias/genética , Cardiomiopatias/fisiopatologia , Modelos Animais de Doenças , Feminino , Testes de Função Cardíaca/efeitos dos fármacos , Humanos , Masculino , Camundongos , Oligonucleotídeos Antissenso/farmacologia , Agregados Proteicos/efeitos dos fármacos , Resultado do TratamentoRESUMO
Enhancing the functional uptake of antisense oligonucleotide (ASO) in the muscle will be beneficial for developing ASO therapeutics targeting genes expressed in the muscle. We hypothesized that improving albumin binding will facilitate traversal of ASO from the blood compartment to the interstitium of the muscle tissues to enhance ASO functional uptake. We synthesized structurally diverse saturated and unsaturated fatty acid conjugated ASOs with a range of hydrophobicity. The binding affinity of ASO fatty acid conjugates to plasma proteins improved with fatty acid chain length and highest binding affinity was observed with ASO conjugates containing fatty acid chain length from 16 to 22 carbons. The degree of unsaturation or conformation of double bond appears to have no influence on protein binding or activity of ASO fatty acid conjugates. Activity of fatty acid ASO conjugates correlated with the affinity to albumin and the tightest albumin binder exhibited the highest activity improvement in muscle. Palmitic acid conjugation increases ASO plasma Cmax and improved delivery of ASO to interstitial space of mouse muscle. Conjugation of palmitic acid improved potency of DMPK, Cav3, CD36 and Malat-1 ASOs (3- to 7-fold) in mouse muscle. Our approach provides a foundation for developing more effective therapeutic ASOs for muscle disorders.
Assuntos
Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacocinética , Ácido Palmítico/química , Animais , Proteínas Sanguíneas/metabolismo , Antígenos CD36/genética , Caveolina 3/genética , Ácidos Graxos/química , Ácidos Graxos Insaturados/química , Masculino , Camundongos Endogâmicos C57BL , Miotonina Proteína Quinase/genética , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/metabolismo , RNA Longo não Codificante/metabolismo , Relação Estrutura-AtividadeRESUMO
Chemerin is a contractile adipokine, produced in liver and fat, and removal of the protein by antisense oligonucleotides (ASO) lowers blood pressure in the normal Sprague Dawley rat. In humans, chemerin is positively associated with blood pressure and obesity so we hypothesized that in a model of hypertension derived from high-fat (HF) feeding, the chemerin ASO would reduce blood pressure more than a high-salt (HS) model. Male Dahl S rats were given a HF (60% kcal fat; age 3-24 wk) or HS diet (4% salt; age 20-24 wk to match age and blood pressure of HF animals). Scrambled control, whole body, or liver-specific ASOs that knock down chemerin were delivered subcutaneously once per week for 4 wk with tissue and blood collected 2 days after the last injection. Conscious blood pressure was measured 24 h/day by radiotelemetry. By the end of whole body ASO administration, blood pressure of HF animals had fallen 29 ± 2 mmHg below baseline, while blood pressure of HS-diet animals fell by only 12 ± 4 mmHg below baseline. Administration of a liver-specific ASO to HF Dahl S resulted in a 6 ± 2 mmHg fall in blood pressure below baseline. Successful knockdown of chemerin in both the whole body and liver-specific administration was confirmed by Western and PCR. These results suggest that chemerin, not derived from liver but potentially from adipose tissue, is an important driver of hypertension associated with high fat. This knowledge could lead to the development of antihypertensive treatments specifically targeted to obesity-associated hypertension.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Quimiocinas/antagonistas & inibidores , Gorduras na Dieta/farmacologia , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/antagonistas & inibidores , Cloreto de Sódio na Dieta/farmacologia , Tecido Adiposo/metabolismo , Animais , Quimiocinas/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Hipertensão/complicações , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Obesidade/complicações , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Ratos , Ratos Endogâmicos DahlRESUMO
Chemerin is an inflammatory adipokine positively associated with hypertension and obesity. The majority of chemerin derives from the liver and adipose tissue, however, their individual contributions to blood pressure are unknown. We began studying chemerin in the normal rat using antisense oligonucleotides (ASO) with whole-body activity (Gen 2.5 chemerin ASO) or liver-restricted activity (GalNAc chemerin ASO). We hypothesized that in normotensive male Sprague-Dawley rats, circulating chemerin is predominately liver-derived and regulates blood pressure. A dosing study of the Gen 2.5 chemerin ASO (with a scrambled control ASO) supported 25 mg/kg as the appropriate dose. GalNAc chemerin ASO was also assessed and used at 10 mg/kg. Radiotelemetry monitored mean arterial pressure (MAP) for a 1-week baseline and weekly subcutaneous ASO injections for 4 weeks. Two days after the final injection, animals were euthanized for tissue reverse transcription-polymerase chain reaction and chemerin Western analysis. Gen 2.5 chemerin ASO treatments reduced chemerin mRNA and protein in liver, retroperitoneal fat (RP), and mesenteric perivascular adipose tissue (mPVAT), as well as reducing protein in plasma. GalNAc chemerin ASO treatments reduced chemerin mRNA and protein in liver and chemerin protein in plasma but had no effect on expression in RP fat or mPVAT. Gen 2.5 chemerin ASO treatment reduced MAP compared with control ASO but was unchanged in animals receiving the GalNAc chemerin ASO. Although circulating chemerin is liver-derived, it does not play a major role in blood pressure regulation. Local effects of chemerin from fat may explain this discrepancy and support chemerin's association with hypertension and obesity.
Assuntos
Pressão Sanguínea/genética , Quimiocinas/deficiência , Quimiocinas/genética , Técnicas de Silenciamento de Genes , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Fígado/metabolismo , Oligonucleotídeos Antissenso/genética , Animais , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Activation of the intrarenal renin angiotensin system (RAS) is believed to play an important role in the development of hypertension and cystogenesis in autosomal dominant polycystic kidney disease (ADPKD). Results of clinical studies testing RAS inhibitors in slowing the progression of cystic disease in ADPKD are inconclusive, and we hypothesized that current RAS inhibitors do not adequately suppress intrarenal RAS. For this study, we compared a novel Gen 2 antisense oligonucleotide (ASO) that inhibits angiotensinogen (Agt) synthesis to lisinopril in adult conditional Pkd1 systemic-knockout mice, a model of ADPKD. Six weeks after Pkd1 global gene knockout, the mice were treated with Agt-ASO (66 mg/kg/wk), lisinopril (100 mg/kg/d), PBS (control), or scrambled ASO (66 mg/kg/wk) for 10 wk, followed by tissue collection. Agt ASO resulted in significant reduction in plasma, liver, and kidney Agt, and increased kidney renin compared with control treatments. Kidneys from Agt-ASO-treated mice were not as enlarged and showed reduced cystic volume compared with lisinopril or control treatments. Blood pressure was better controlled with lisinopril than with Agt-ASO. Agt-ASO suppressed cell proliferation in both cystic and noncystic cells compared with lisinopril and control treatments. However, Agt-ASO did not reduce cell proliferation in liver, which indicates that Agt-ASO targets cell signaling pathways that specifically suppresses cystogenesis in the kidney. These data suggest that Agt-ASO effectively attenuates intrarenal RAS and therefore can be a novel and effective agent for treating ADPKD.
Assuntos
Angiotensinogênio/metabolismo , Doenças Renais Policísticas/metabolismo , Canais de Cátion TRPP/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Angiotensinogênio/biossíntese , Animais , Feminino , Rim/efeitos dos fármacos , Rim/metabolismo , Lisinopril/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doenças Renais Policísticas/genética , Canais de Cátion TRPP/genéticaRESUMO
Mitochondrial electron transport chain (ETC) is the major source of reactive oxygen species during myocardial ischemia-reperfusion (I/R) injury. Ischemic defect and reperfusion-induced injury to ETC are critical in the disease pathogenesis of postischemic heart. The properties of ETC were investigated in an isolated heart model of global I/R. Rat hearts were subjected to ischemia for 30 min followed by reperfusion for 1 h. Studies of mitochondrial function indicated a biphasic modulation of electron transfer activity (ETA) and ETC protein expression during I/R. Analysis of ETAs in the isolated mitochondria indicated that complexes I, II, III, and IV activities were diminished after 30 min of ischemia but increased upon restoration of flow. Immunoblotting analysis and ultrastructural analysis with transmission electron microscopy further revealed marked downregulation of ETC in the ischemic heart and then upregulation of ETC upon reperfusion. No significant difference in the mRNA expression level of ETC was detected between ischemic and postischemic hearts. However, reperfusion-induced ETC biosynthesis in myocardium can be inhibited by cycloheximide, indicating the involvement of translational control. Immunoblotting analysis of tissue homogenates revealed a similar profile in peroxisome proliferator-activated receptor-γ coactivator-1α expression, suggesting its essential role as an upstream regulator in controlling ETC biosynthesis during I/R. Significant impairment caused by ischemic and postischemic injury was observed in the complexes I- III. Analysis of NADH ferricyanide reductase activity indicated that injury of flavoprotein subcomplex accounts for 50% decline of intact complex I activity from ischemic heart. Taken together, our findings provide a new insight into the molecular mechanism of I/R-induced mitochondrial dysfunction.
Assuntos
Transporte de Elétrons/fisiologia , Mitocôndrias Cardíacas/fisiologia , Isquemia Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Difosfato de Adenosina/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Western Blotting , Circulação Coronária/efeitos dos fármacos , Circulação Coronária/fisiologia , Cicloeximida/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Radicais Livres/metabolismo , Frequência Cardíaca/fisiologia , Técnicas In Vitro , Masculino , Microscopia Eletrônica de Transmissão , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/enzimologia , NADH Desidrogenase/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Função Ventricular Esquerda/fisiologiaRESUMO
We evaluated the potential of AGTR1, the principal receptor for angiotensin II (Ang II) and a member of the G protein-coupled receptor family, for targeted delivery of antisense oligonucleotides (ASOs) in cells and tissues with abundant AGTR1 expression. Ang II peptide ASO conjugates maintained robust AGTR1 signaling and receptor internalization when ASO was placed at the N-terminus of the peptide, but not at C-terminus. Conjugation of Ang II peptide improved ASO potency up to 12- to 17-fold in AGTR1-expressing cells. Additionally, evaluation of Ang II conjugates in cells lacking AGTR1 revealed no enhancement of ASO potency. Ang II peptide conjugation improves potency of ASO in mouse heart, adrenal, and adipose tissues. The data presented in this report add to a growing list of approaches for improving ASO potency in extrahepatic tissues.
Assuntos
Oligonucleotídeos Antissenso , Receptor Tipo 1 de Angiotensina , Animais , Camundongos , Oligonucleotídeos Antissenso/farmacologia , Receptor Tipo 1 de Angiotensina/genética , Transdução de SinaisRESUMO
Immune cell invasion after the transplantation of solid organs is directed by chemokines binding to glycosaminoglycans (GAGs), creating gradients that guide immune cell infiltration. Renal transplant is the preferred treatment for end stage renal failure, but organ supply is limited and allografts are often injured during transport, surgery or by cytokine storm in deceased donors. While treatment for adaptive immune responses during rejection is excellent, treatment for early inflammatory damage is less effective. Viruses have developed highly active chemokine inhibitors as a means to evade host responses. The myxoma virus-derived M-T7 protein blocks chemokine: GAG binding. We have investigated M-T7 and also antisense (ASO) as pre-treatments to modify chemokine: GAG interactions to reduce donor organ damage. Immediate pre-treatment of donor kidneys with M-T7 to block chemokine: GAG binding significantly reduced the inflammation and scarring in subcapsular and subcutaneous allografts. Antisense to N-deacetylase N-sulfotransferase1 (ASONdst1) that modifies heparan sulfate, was less effective with immediate pre-treatment, but reduced scarring and C4d staining with donor pre-treatment for 7 days before transplantation. Grafts with conditional Ndst1 deficiency had reduced inflammation. Local inhibition of chemokine: GAG binding in donor organs immediately prior to transplant provides a new approach to reduce transplant damage and graft loss.
RESUMO
Heart failure (HF) is a major cause of morbidity and mortality worldwide, highlighting an urgent need for novel treatment options, despite recent improvements. Aberrant Ca2+ handling is a key feature of HF pathophysiology. Restoring the Ca2+ regulating machinery is an attractive therapeutic strategy supported by genetic and pharmacological proof of concept studies. Here, we study antisense oligonucleotides (ASOs) as a therapeutic modality, interfering with the PLN/SERCA2a interaction by targeting Pln mRNA for downregulation in the heart of murine HF models. Mice harboring the PLN R14del pathogenic variant recapitulate the human dilated cardiomyopathy (DCM) phenotype; subcutaneous administration of PLN-ASO prevents PLN protein aggregation, cardiac dysfunction, and leads to a 3-fold increase in survival rate. In another genetic DCM mouse model, unrelated to PLN (Cspr3/Mlp-/-), PLN-ASO also reverses the HF phenotype. Finally, in rats with myocardial infarction, PLN-ASO treatment prevents progression of left ventricular dilatation and improves left ventricular contractility. Thus, our data establish that antisense inhibition of PLN is an effective strategy in preclinical models of genetic cardiomyopathy as well as ischemia driven HF.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Cardiomiopatias/genética , Cardiomiopatias/terapia , Terapia Genética , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/terapia , Oligonucleotídeos Antissenso/genética , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cardiomiopatias/metabolismo , Feminino , Insuficiência Cardíaca/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Ratos , Ratos Endogâmicos LewRESUMO
Extracting and concentrating mitochondrial protein complexes from gel strips after blue native PAGE (BN-PAGE) can be daunting tasks using the traditional methods, such as electroelution, passive diffusion and centrifugal concentration. We present a simplified gel electrophoresis method to concentrate mitochondrial protein complexes with excellent recovery rate. Mitochondrial complex I present in a long gel strip from BN-PAGE can be easily concentrated into a 0.8 cm gel strip when a second BN-PAGE is performed with a Y-shaped gel and the addition of 0.01% n-dodecyl beta-D-maltoside and 0.001% SDS in the cathode buffer. Once completed, the concentrated protein complex in the gel strip is ready for SDS-PAGE or proteomic studies.
Assuntos
Complexo I de Transporte de Elétrons/isolamento & purificação , Eletroforese em Gel de Poliacrilamida/métodos , Membranas Mitocondriais/química , Proteínas Mitocondriais/química , Animais , Complexo I de Transporte de Elétrons/química , Glucosídeos/química , Ratos , Dodecilsulfato de Sódio/químicaRESUMO
During cardiac arrest (CA), myocardial perfusion is solely dependent on cardiopulmonary resuscitation (CPR) although closed-chest compressions only provide about 10-20% of normal myocardial perfusion. The study was conducted in a whole animal CPR model to determine whether CPR-generated oxygen delivery preserves or worsens mitochondrial function. Male Sprague-Dawley rats (400-450 g) were randomly divided into four groups: (1) BL (instrumentation only, no cardiac arrest), (2) CA(15) (15 min cardiac arrest without CPR), (3) CA(25) (25 min cardiac arrest without CPR) and (4) CPR (15 min cardiac arrest, followed by 10 min CPR). The differences between groups were evaluated by measuring mitochondrial respiration, electron transport chain (ETC) complex activities and mitochondrial ultrastructure by transmission electron microscopy (TEM). The CA(25) group had the greatest impairment of mitochondrial respiration and ETC complex activities (I-III). In contrast, the CPR group was not different from the CA(15) group regarding all measures of mitochondrial function. Complex I was more susceptible to ischemic injury than the other complexes and was the major determinant of mitochondrial dysfunction. Observations of mitochondrial ultrastructure by TEM were compatible with the biochemical results. The findings suggest that, despite low blood flow and oxygen delivery, CPR is able to preserve heart mitochondrial function and viability during ongoing global ischemia. Preservation of complex I activity and mitochondrial function during cardiac arrest may be an important mechanism underlying the beneficial effects of CPR which have been shown in clinical studies.
Assuntos
Reanimação Cardiopulmonar , Parada Cardíaca/fisiopatologia , Mitocôndrias Cardíacas/metabolismo , Animais , Western Blotting , Respiração Celular , Citocromos a/metabolismo , Densitometria , Transporte de Elétrons , Parada Cardíaca/patologia , Masculino , Mitocôndrias Cardíacas/ultraestrutura , Ratos , Ratos Sprague-DawleyRESUMO
Movement of circulating fatty acids (FAs) to parenchymal cells requires their transfer across the endothelial cell (EC) barrier. The multiligand receptor cluster of differentiation 36 (CD36) facilitates tissue FA uptake and is expressed in ECs and parenchymal cells such as myocytes and adipocytes. Whether tissue uptake of FAs is dependent on EC or parenchymal cell CD36, or both, is unknown. Using a cell-specific deletion approach, we show that EC, but not parenchymal cell, CD36 deletion increased fasting plasma FAs and postprandial triglycerides. EC-Cd36-KO mice had reduced uptake of radiolabeled long-chain FAs into heart, skeletal muscle, and brown adipose tissue; these uptake studies were replicated using [11C]palmitate PET scans. High-fat diet-fed EC-CD36-deficient mice had improved glucose tolerance and insulin sensitivity. Both EC and cardiomyocyte (CM) deletion of CD36 reduced heart lipid droplet accumulation after fasting, but CM deletion did not affect heart glucose or FA uptake. Expression in the heart of several genes modulating glucose metabolism and insulin action increased with EC-CD36 deletion but decreased with CM deletion. In conclusion, EC CD36 acts as a gatekeeper for parenchymal cell FA uptake, with important downstream effects on glucose utilization and insulin action.
Assuntos
Antígenos CD36/metabolismo , Células Endoteliais/metabolismo , Ácidos Graxos/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Transporte Biológico Ativo/genética , Antígenos CD36/genética , Células Endoteliais/patologia , Ácidos Graxos/genética , Glucose/genética , Glucose/metabolismo , Humanos , Resistência à Insulina , Camundongos , Camundongos Knockout , Miocárdio/patologia , Miócitos Cardíacos/patologia , Especificidade de ÓrgãosRESUMO
Uncontrolled hypertension is an important contributor to cardiovascular disease. Despite the armamentarium of antihypertensive treatments, there remains a need for novel agents effective in individuals who cannot reach acceptable blood pressure levels. Inhibitors targeting the renin-angiotensin-aldosterone system (RAAS) are widely used but may not optimally inhibit RAAS and demonstrate an acceptable safety profile. Experiments were conducted to characterize a series of AGT (angiotensinogen) antisense oligonucleotides (ASOs) and compare their efficacy and tolerability to traditional RAAS blockade. AGT ASOs which target multiple systemic sites of AGT versus an N-acetylgalactosamine-conjugated AGT ASO that targets the liver were compared with captopril and losartan. Spontaneously hypertensive rats fed an 8% NaCl diet, a model of malignant hypertension resistant to standard RAAS inhibitors, demonstrated robust and durable blood pressure reductions with AGT ASO treatments, which was not observed with standard RAAS blockade. Studies in rat models of acute kidney injury produced by salt deprivation revealed kidney injury with ASO treatment that reduced kidney-expressed AGT, but not in animals treated with the N-acetylgalactosamine AGT ASO despite comparable plasma AGT reductions. Administration of either captopril or losartan also produced acute kidney injury during salt deprivation. Thus, intrarenal RAAS derived from kidney AGT, and inhibited by the standard of care, contributes to the maintenance of renal function during severe RAAS challenge. Such improvements in efficacy and tolerability by a liver-selective AGT inhibitor could be desirable in individuals not at their blood pressure goal with existing RAAS blockade.
Assuntos
Injúria Renal Aguda , Angiotensinogênio/metabolismo , Hipertensão , Oligonucleotídeos Antissenso , Sistema Renina-Angiotensina , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/fisiopatologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Modelos Animais de Doenças , Resistência a Medicamentos/fisiologia , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Rim/metabolismo , Rim/fisiopatologia , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Ratos , Ratos Endogâmicos SHR , Sistema Renina-Angiotensina/efeitos dos fármacos , Sistema Renina-Angiotensina/fisiologia , Resultado do TratamentoRESUMO
Preclinical and clinical data suggest CD40 activation contributes to renal inflammation and injury. We sought to test whether upregulation of CD40 in the kidney is a causative factor of renal pathology and if reduction of renal CD40 expression, using antisense oligonucleotides (ASOs) targeting CD40, would be beneficial in mouse models of glomerular injury and unilateral ureter obstruction. Administration of a Generation 2.5 CD40 ASO reduced CD40 mRNA and protein levels 75-90% in the kidney. CD40 ASO treatment mitigated functional, transcriptional, and pathological endpoints of doxorubicin-induced nephropathy. Experiments using an activating CD40 antibody revealed CD40 is primed in kidneys following doxorubicin injury or unilateral ureter obstruction and CD40 ASO treatment blunted CD40-dependent renal inflammation. Suborgan fractionation and imaging studies demonstrated CD40 in glomeruli before and after doxorubicin administration that becomes highly enriched within interstitial and glomerular foci following CD40 activation. Such foci were also sites of ASO distribution and activity and may be predominately comprised from myeloid cells as bone marrow CD40 deficiency sharply attenuated CD40 antibody responses. These studies suggest an important role of interstitial renal and/or glomerular CD40 to augment kidney injury and inflammation and demonstrate that ASO treatment could be an effective therapy in such disorders.
RESUMO
We recently showed that Bendavia, a novel mitochondria-targeting peptide, reduced infarction and no-reflow across several experimental models. The purpose of this study was to determine the therapeutic timing and mechanism of action that underlie Bendavia's cytoprotective property. In rabbits exposed to in vivo ischemia/reperfusion (30/180 min), Bendavia administered 20 minutes prior to reperfusion (0.05 mg/kg/h, intravenously) reduced myocardial infarct size by â¼50% when administered for either 1 or 3 hours of reperfusion. However, when Bendavia perfusion began just 10 minutes after the onset of reperfusion, the protection against infarction and no-reflow was completely lost, indicating that the mechanism of protection is occurring early in reperfusion. Experiments in isolated mouse liver mitochondria found no discernible effect of Bendavia on blocking the permeability transition pore, and studies in isolated heart mitochondria showed no effect of Bendavia on respiratory rates. As Bendavia significantly lowered reactive oxygen species (ROS) levels in isolated heart mitochondria, the ROS-scavenging capacity of Bendavia was compared to well-known ROS scavengers using in vitro (cell-free) systems that enzymatically generate ROS. Across doses ranging from 1 nmol/L to 1 mmol/L, Bendavia showed no discernible ROS-scavenging properties, clearly differentiating itself from prototypical scavengers. In conclusion, Bendavia is a promising candidate to reduce cardiac injury when present at the onset of reperfusion but not after reperfusion has already commenced. Given that both infarction and no-reflow are related to increased cellular ROS, Bendavia's protective mechanism of action likely involves reduced ROS generation (as opposed to augmented scavenging) by endothelial and myocyte mitochondria.
Assuntos
Mitocôndrias Hepáticas/efeitos dos fármacos , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Oligopeptídeos/farmacologia , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sequestradores de Radicais Livres/administração & dosagem , Sequestradores de Radicais Livres/farmacologia , Cobaias , Masculino , Camundongos , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Oligopeptídeos/administração & dosagem , Coelhos , Espécies Reativas de Oxigênio/metabolismo , Fatores de TempoRESUMO
BACKGROUND: The effect of hyperoxygenation at reperfusion, particularly in the setting of cardiac arrest, remains unclear. This issue was studied in a prolonged cardiac arrest model consisting of 25 min cardiac arrest in a rat resuscitated with cardiopulmonary bypass (CPB). The objective of this study was to determine the effect of hyperoxygenation following prolonged cardiac arrest resuscitation on mitochondrial and cardiac function. METHODS: Male Sprague-Dawley rats (400-450 g) were anesthetized with ketamine and xylazine and instrumented for closed chest cardiopulmonary bypass (CPB). Following a 25-min KCl-induced cardiac arrest, the animals were resuscitated by CPB with 100% oxygen. Three minutes after successful return of spontaneous circulation (ROSC), the animals received either normoxemic reperfusion (CPB with 40-50% oxygen) or hyperoxemic reperfusion (CPB with 100% oxygen) for 1 h. Post-resuscitation hemodynamics, cardiac function, mitochondrial function and immunostaining of 3-nitrotyrosine were compared between the two different treatment groups. RESULTS: At 1 h after ROSC, the hyperoxemic reperfusion group had a significant higher mean arterial pressure, less metabolic acidosis and better diastolic function than the normoxemic reperfusion group. Cardiac mitochondria from the hyperoxemic reperfusion group had a higher respiratory control ratio (RCR) and cardiac tissue showed less nitroxidative stress compared to the normoxemic reperfusion group. CONCLUSIONS: One hour of hyperoxemic reperfusion after 25 min of cardiac arrest in an in vivo CPB model resulted in significant short-term improvement in myocardial and mitochondrial function compared with 1h of normoxemic reperfusion. This myocardial response may differ from previously reported post-arrest hyperoxia mediated effects following shorter arrest times.
Assuntos
Ponte Cardiopulmonar/métodos , Parada Cardíaca/terapia , Hiperóxia , Oxigenoterapia/métodos , Análise de Variância , Animais , Gasometria , Parada Cardíaca/fisiopatologia , Hemodinâmica , Imuno-Histoquímica , Masculino , Mitocôndrias Cardíacas/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Tempo , Tirosina/análogos & derivados , Tirosina/análiseRESUMO
The neutrophil elastase inhibitor sivelestat (ONO-5046) possesses unknown mechanisms of cardioprotection when infused following global ischemia, even in the absence of neutrophils. Since myocardial ischemia-reperfusion injury is strongly associated with endothelial dysfunction and reactive oxygen species (ROS) generation during reperfusion, we have tested the hypothesis that infusion of sivelestat during postischemic low flow would preserve endothelial and contractile function and reduce infarct size through an ROS-mediated mechanism. Isolated male rat hearts, subjected to global ischemia of 25 minutes, were reperfused with low flow with or without sivelestat followed by a full flow reperfusion. Hearts treated with sivelestat showed a significant improvement of LV contractile function and a reduction in infarct size. Infusion of L-NAME (nonspecific blocker of endothelial nitric oxide synthase (eNOS)) along with sivelestat during reperfusion reversed the preservation of contractile function and infarct size. In vitro EPR spin trapping experiments showed that sivelestat treatment decreased superoxide adduct formation in bovine aortic endothelial cells (BAECs) subjected to hypoxia-reoxygenation. Similarly, dihydroethidine (DHE) staining showed decreased superoxide production in LV sections from sivelestat-treated hearts. Taken together, these results indicate that sivelestat infusion during postischemic low flow reduces infarct size and preserves vasoreactivity in association with decreased ROS formation and the preservation of nitric oxide.
Assuntos
Circulação Coronária/efeitos dos fármacos , Glicina/análogos & derivados , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Sulfonamidas/administração & dosagem , Sulfonamidas/uso terapêutico , Animais , Aorta/patologia , Cardiotônicos/farmacologia , Cardiotônicos/uso terapêutico , Bovinos , Creatina Quinase/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Glicina/administração & dosagem , Glicina/farmacologia , Glicina/uso terapêutico , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Técnicas In Vitro , Masculino , Modelos Cardiovasculares , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/enzimologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Sulfonamidas/farmacologia , Superóxidos/metabolismo , Vasoconstrição/efeitos dos fármacos , Função Ventricular Esquerda/efeitos dos fármacosRESUMO
INTRODUCTION: Rapid post-ischemic re-oxygenation is necessary to minimize ischemic injury, but itself can induce further reperfusion injury through the induction of reactive oxygen species. Utilization of oxygen within the cell primarily occurs in the mitochondria. The objective of this study was to determine heart mitochondrial function after 1 h of controlled arterial oxygenation following cardiac arrest and restoration of spontaneous circulation (ROSC). We hypothesized that arterial hyper-oxygenation following ROSC would result in greater impairment of heart mitochondrial function. METHODS: KCl cardiac arrest was induced in anesthetized rats. Following 6.5 min of cardiac arrest, animals were resuscitated with standard thumper CPR, ventilation and epinephrine. Following ROSC, all animals were ventilated for 60 min with either 100% O(2) or 40% O(2) titrated to achieve normoxia utilizing pulse oximetry. At the end of 1 h, heart mitochondria were isolated and mitochondrial respiratory function was measured. RESULTS: Post-ROSC arterial PaO2 was 280 ± 40 in the 100% O2 group and 105 ± 10 in the 40% O2 group. One hour after ROSC, heart mitochondrial state 3 respirations and respiration control ratio (state 3/4 respiration) were significantly reduced from baseline in animals ventilated with 100% O(2), but not with 40% O(2). CONCLUSION: Post-ROSC arterial hyperoxia after a short cardiac arrest exacerbates impaired mitochondrial function. The overall clinical significance of these findings is unclear and requires additional work to better understand the role of post-arrest hyperoxia on cardiac and mitochondrial function.
Assuntos
Parada Cardíaca/complicações , Hiperóxia/etiologia , Mitocôndrias Cardíacas/fisiologia , Traumatismo por Reperfusão Miocárdica/etiologia , Oxigênio/metabolismo , Animais , Reanimação Cardiopulmonar , Modelos Animais de Doenças , Parada Cardíaca/fisiopatologia , Parada Cardíaca/terapia , Hiperóxia/metabolismo , Hiperóxia/fisiopatologia , Masculino , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Consumo de Oxigênio , Ratos , Ratos Sprague-DawleyRESUMO
AIM OF STUDY: Isolated hearts used in the study of ischemia-reperfusion induced myocardial reactive oxygen species (ROS) have typically been perfused with crystalloid buffer. Limitations of crystalloid buffer which may exaggerate the production of ROS, include a requirement for higher oxygen tension and the absence of the intrinsic erythrocyte antioxidant defenses. Using a novel recirculating blood-perfused rat heart model, we measured H(2)O(2) concentration in the blood (as an indicator of ROS formation) and tissue glutathione concentration (an overall measure of oxidant stress) following ischemia and reperfusion. METHODS: Autologous blood was obtained and the heart isolated from pentobarbital-anesthetized male Sprague-Dawley rats and placed on a recirculating perfusion circuit with an in-line peristaltic pump and oxygenator. Blood temperature was maintained at 37°C. Hearts underwent normal perfusion for 120min (Sham Group, n=7) or 35min of normal perfusion, 25min of global ischemia, followed by 60min of reperfusion with baseline coronary blood flow levels (IR group, n=6). Oxygen delivery was compared with a group of buffer-perfused hearts perfused at 85mmHg. RESULTS: LV function in the sham group remained stable for 2h under normal physiologic oxygen conditions. The oxygen tension and coronary flow were significantly decreased but the myocardial oxygen delivery was significantly increased with blood perfusion compared with buffer perfusion. In the blood IR group, a significant increase in H(2)O(2) was seen early in reperfusion and a reduction in tissue GSH was noted at the end of reperfusion. CONCLUSION: This model offers significant physiologic advantages in the study of ischemia and reperfusion, particularly in terms of oxygen delivery, compared with the more commonly used acellular buffer-perfused isolated heart systems.
Assuntos
Peróxido de Hidrogênio/sangue , Reperfusão Miocárdica , Estresse Oxidativo , Animais , Glutationa/metabolismo , Técnicas In Vitro , Masculino , Modelos Animais , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Recent scientific evidence has demonstrated the importance of good quality chest compressions without interruption to improve cardiac arrest resuscitation rates, and suggested that a de-emphasis on minute ventilation is needed. However, independent of ventilation, the role of oxygen and the optimal oxygen concentration during CPR is not known. Previous studies have shown that ventilation with high oxygen concentration after CPR is associated with worse neurologic outcome. We tested the hypothesis that initial ventilation during CPR without oxygen improves resuscitation success. METHODS: Sprague-Dawley rats were anesthetized with ketamine/xylazine (IP), intubated and ventilated with room air. A KCl bolus (0.04 mg/g) was given (IV) to induce asystolic cardiac arrest and ventilation was stopped. At 6 min, CPR was started with an automated chest compressor at a rate of 200-240/min and epinephrine (0.01 mg/kg) was given 1 min later. During CPR, the ventilation rate was 50% of baseline with one of three oxygen concentrations: (1) 0% O2 (100% N2), (2) 21% O2, or (3) 100% O2. The prescribed oxygen concentration was continued for 2 min after return of spontaneous circulation (ROSC) and then all animals were switched to 100% oxygen for 1h prior to extubation. Blood gases were measured at baseline, 2 min and 1h after ROSC. Group comparisons were done using Fisher's exact test and ANOVA. RESULTS: ROSC was achieved in 1/10 (0% O2), 9/11 (21% O2) and 10/12 (100% O2, p<0.001). ROSC times after starting CPR were 80s in the 0% O2, 115+/-87 s in the 21% O2 group and 95+/-33 s in the 100% O2 group (mean+/-SD, p=0.5). Aortic end-diastolic pressure before ROSC was not different among groups. 100% oxygen ventilation in the first 2 min resulted in higher PaO2 at ROSC 2 min (109+/-44 mm Hg vs. 33+/-8 mm Hg, p<0.001). Survival to 72 h was 0/1 (0% O2), 7/9 (21% O2) and 8/10 (100% O2) with a low neurologic deficit score in both O2 groups (NDS range 5-25). CONCLUSIONS: In a mild cardiac arrest model with generally good neurologic recovery, initial CPR ventilation with no O2 did not allow for ROSC. In contrast, CPR coupled with room air or higher oxygen levels result in a high rate of ROSC with good neurologic recovery. During CPR, the level of oxygenation must be considered, which if too low may preclude initial ROSC.