RESUMEN
OBJECTIVE: In this study, experiments were designed to determine which parts and how carvedilol provided protection of the failed hearts under cardioplegia-induced hypoxia/reperfusion (H/R) insult. BACKGROUND: Carvedilol could improve the perioperative and postoperative prognosis of the heart failure patients. The mechanism of carvedilol in prevention of the occurrence of cardiomyocytic apoptosis and preservation of cardiac function had been rarely studied. METHODS: Fifty Sprague-Dawley rats treated with placebo or daily carvedilol after coronary artery ligation were divided into five groups (ligation only-group 2; ligation followed with cardioplegia-induced cardiac arrest-group 3; daily oral carvedilol treatment at 0.1, 1.0, or 10.0 mg/kg after ligation followed with cardioplegia-induced cardiac arrest-groups 4, 5, and 6), whereas another 10 animals received sham operation only (group 1), 30 days before ex vivo H/R injury. Failed hearts were harvested and received 1-h cardiac hypoxia with intermittent cold cardioplegia infusion and followed by 2-h reperfusion with warm oxygenated phosphate-buffered saline solution using a Langendorff apparatus. Perfusate was sampled at various time points. After H/R injury, the myocardium was carefully dissected into infarct, peri-infarct, and remote zones, which were used for further studies. In vitro H/R studies were carried on HL-1 cardiomyocytes to further explore the possible downstream pathways. RESULTS: Carvedilol could reduce the H/R-induced cardiomyocytic apoptosis and related proteins expression, especially in the peri-infarct zone, in a dose-dependent pattern. Carvedilol could also preserve cardiac contractility via modulation of the tumor necrosis factor α and interleukin 8 mRNA expression and reduction of Bcl-2 and cytochrome c protein production and decrease the occurrence of apoptosis ex vivo. In vitro experiments revealed that carvedilol exerted its antiapoptotic effect by way of phosphatidylinositol 3-kinase and MEK (mitogen-activated protein-kinase kinase), instead of protein kinase A, pathways. CONCLUSIONS: In the failed heart, pretreatment with carvedilol could preserve cardiac contractility during cardioplegia-induced myocardial H/R injury by lessening inflammation-related genes and expression of cytokines, decreasing apoptosis-related proteins production and diminishing the occurrence of cardiomyocytic apoptosis in the peri-infarct zone. The cardinal pathways of the antiapoptotic mechanism of carvedilol were PI3K- and MEK-related pathways.
Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Apoptosis/efectos de los fármacos , Carbazoles/farmacología , Infarto del Miocardio/tratamiento farmacológico , Miocitos Cardíacos/patología , Propanolaminas/farmacología , Animales , Biomarcadores/metabolismo , Carvedilol , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Paro Cardíaco Inducido , Ligadura , Masculino , Infarto del Miocardio/patología , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/prevención & control , Ratas , Ratas Sprague-DawleyRESUMEN
In this study, experiments were designed to determine whether microRNAs (miRNAs) play a role in the regulation or modulation of cardiomyocytic reactions under cardioplegia-induced cardiac arrest during cardiopulmonary bypass. MicroRNAs play powerful and unexpected roles in numerous cardiovascular diseases. MicroRNA-based therapeutics may provide a unique opportunity to translate this knowledge into the clinical setting. Sprague-Dawley rats (10 per group) were randomly divided into three groups: control, perfusion, and arrest groups. In the perfusion group, isolated hearts were perfused with oxygenated physiologic buffered solution for 3 h using a Langendorff apparatus. In the arrest group, cold crystalloid cardioplegia solution was used to induce and maintain cardiac arrest for 1 h; hearts were reperfused for 2 h with warm oxygenated phosphate-buffered saline solution. Cardiac miRNAs and protein expression patterns were detected using miRNA arrays and two-dimensional fluorescence difference gel electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Of 103 different miRNAs and 222 different proteins expressed in the three groups, miRNA-27a was the one considered to be related to the regulation of cardiomyocyte apoptosis by targeting the interleukin 10 pathway. Transfection of H9c2 cardiomyocytes with pre-miRNA-27a, which significantly decreased the mRNA and protein levels of interleukin 10 and increased expression of nuclear factor κB and its downstream cytokines during hypoxia/reperfusion injury, could activate caspase 3 and apoptosis. Our study demonstrated the altered expression of miRNAs in cardiomyocytes during cardioplegia-induced cardiac arrest. The involvement of miRNAs in cardiomyocytic apoptosis adds another level of complexity to gene regulation, which could open up novel avenues for cardiac protection strategies during cardiac surgery.
Asunto(s)
Apoptosis , Paro Cardíaco Inducido , Interleucina-10/biosíntesis , MicroARNs/metabolismo , Proteínas Musculares/biosíntesis , Miocitos Cardíacos/metabolismo , Transducción de Señal , Animales , Caspasa 3/genética , Caspasa 3/metabolismo , Línea Celular , Interleucina-10/genética , MicroARNs/genética , Proteínas Musculares/genética , Miocitos Cardíacos/patología , FN-kappa B/genética , FN-kappa B/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , TransfecciónRESUMEN
BACKGROUND: Our previous studies revealed that cardioplegia-induced cardiac arrest under cardiopulmonary bypass (CPB) decreased cardiomyocytic nitric oxide and increased apoptosis. We hypothesized that pretreatment with bradykinin (BK) would improve the profile of anti-apoptotic proteins and inhibit cardiomyocytic apoptosis. MATERIALS AND METHODS: New Zealand white rabbits received total CPB. Rabbits were weaned from CPB and reperfused for 4 h. Blood was sampled at various time points. Bradykinin and/or nitric oxide synthase (NOS) inhibitors or BK-receptor antagonists were infused systemically 30 min before beginning of CPB, and continued throughout the procedure. The ascending aorta was cross-clamped for 60 min while cold crystalloid cardioplegic solution was intermittently infused into the aortic root. The hearts were harvested and studied for evidence of apoptosis and ischemia/reperfusion induced inflammation-related cytokine production by cardiomyocytes. RESULTS: Our results revealed that bradykinin supplementation during cardioplegia could prevent I/R-induced inflammatory and apoptotic effects, which could be reversed with a NOS inhibitor. BK antagonists and NOS inhibitors worsened the inflammatory and apoptotic responses of cardiomyocytes, which could be reversed with an exogenous NO donor. CONCLUSIONS: Restoring the NO concentration after cardioplegia-induced cardiac arrest (CCA) under CPB with bradykinin could modulate (1) the nuclear translocation of NF-kappaB, (2) the plasma levels of inflammation-related cytokines, (3) the Bcl-2/Bax ratio, and (4) the occurrence of apoptosis. Exogenous bradykinin administration was associated with the myocardial apoptotic response by inhibition of NF-kappaB translocation, inflammatory cytokine production, Akt activation, and elevation of the Bcl-2/Bax ratio via a NO-mediated pathway.
Asunto(s)
Apoptosis/efectos de los fármacos , Bradiquinina/uso terapéutico , Daño por Reperfusión Miocárdica/prevención & control , Miocitos Cardíacos/efectos de los fármacos , Vasodilatadores/uso terapéutico , Animales , Western Blotting , Bradiquinina/farmacología , Puente Cardiopulmonar , Caspasa 3/metabolismo , Paro Cardíaco Inducido/efectos adversos , Etiquetado Corte-Fin in Situ , Masculino , Daño por Reperfusión Miocárdica/etiología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Miocardio/patología , Miocitos Cardíacos/enzimología , FN-kappa B/metabolismo , Óxido Nítrico/metabolismo , Peroxidasa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Conejos , Troponina I/metabolismo , Factor de Necrosis Tumoral alfa/sangre , Vasodilatadores/farmacología , Función Ventricular Izquierda , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Cardioplegic-induced H/R injury results in cardiomyocytic apoptosis. AMPK has been shown to reduce ER stress and the unfolded protein response (UPR). Whether AMPK activation can attenuate cardiomyocytic apoptosis after cardioplegia-induced H/R injury is unknown. Cardiomyocytes were exposed to simulated ischemia by incubation in a hypoxic chamber with intermittent cold cardioplegia solution infusion at 20-minute intervals and subsequently reoxygenated in a normoxic environment. Various doses of AMPK activators (AICAR or metformin) were given 2 days before H/R injury. The cardiomyocytes were harvested after reoxygenation for subsequent examination. With both AMPK activators, the antiapoptotic genes of ER stress and UPR, the subsequent production of proapoptotic proteins was attenuated, and the antiapoptotic proteins were elevated. The activity of the apoptotic effectors of ER stress was also reduced with AMPK activation. Moreover, TUNEL staining showed that AMPK activation significantly reduced the percentage of apoptotic cardiomyocytes after cardioplegia-induced H/R injury. Our results revealed that AMPK activation during cardioplegia-induced H/R injury attenuates cardiomyocytic apoptosis, via enhancement of antiapoptotic and reduction of proapoptotic responses, resulting from lessening ER stress and the UPR. AMPK activation may serve as a future pharmacological target to reduce H/R injury in the clinical setting.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Apoptosis/fisiología , Retículo Endoplásmico/metabolismo , Hipoxia , Daño por Reperfusión Miocárdica , Miocitos Cardíacos , Estrés Fisiológico , Animales , Biomarcadores/metabolismo , Línea Celular , Activación Enzimática , Humanos , Hipoxia/metabolismo , Hipoxia/patología , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/patología , Miocitos Cardíacos/fisiologíaRESUMEN
GTP cyclohydrolase I (GCH, EC 3.5.4.16) regulates the level of tetrahydrobiopterin and in turn the activities of nitric oxide synthase and aromatic amino acid hydroxylases. Type II GCH mRNA, an alternatively spliced species abundant in blood cells, encodes a truncated and nonfunctional protein. When we stimulate peripheral blood mononuclear cells by PHA, the transcription of full-length GCH mRNA increased, but that of type II mRNA decreased transiently. We further demonstrated that the type II cDNA exerted a dominant-negative effect on the wild-type cDNA, similar to the effect of some GCH mutants. Therefore, type II mRNA may regulate GCH and then contribute to the regulation of NO production by BH4-dependent iNOS in mononuclear cells. Selection of the splicing sites may be coupled with transcriptional activation of the GCH gene.