Presence of autoantibodies against ß1-adrenoceptor aggravates the kidney injury in rats.

Since the autoantibodies against the second extracellular loop of ß(1)-adrenoceptor (ß(1)-AABs) have been found in the sera of patients with idiopathic dilated cardiomyopathy (IDCM), the involvement of autoimmune mechanisms in the pathogenesis of many cardiovascular diseases has extensively been investigated. Our previous study found that urinary occult blood and protein excretion were frequently found in the rats with positive ß(1)-AABs, but the mechanisms are unclear. Therefore, we infused the ß(1)-AABs into the vein periodically in an attempt to investigate whether ß(1)-AABs could induce morphological and functional changes in the kidneys of adult and aged rats and explore the possible mechanisms. The synthetic peptide according to the sequences of the second extracellular loop of ß(1)-adrenoceptor (ß(1)-AR-ECII) was used to immunize the adult rats to acquire enough ß(1)-AABs for use. Neonatal rat ventricular myocytes (NRVMs) culture was used to observe the biological effects of ß(1)-AABs on the beating rate. The purified ß(1)-AABs were transfused into the vein of rats. The sera level of blood urea nitrogen (BUN), creatinine (CR), uric acid (UA), urinary specific gravity, protein excretion, occult blood and urinary glucose were detected at the different time points by biochemistry and urine analyzers. HE and Masson's trichrome staining were used to detect the changes in kidney structure of passively immunized rats. Enhanced green fluorescent protein (EGFP) and ß(1)-AR-EGFP plasmids were transfected into the human embryonic kidney 293 (HEK293) cells in order to observe the changes in cell injury with the treatment of ß(1)-AABs. It was found that the sera level of BUN, CR and UA increased gradually and the ratio of BUN to CR decreased progressively with the administration of ß(1)-AABs. The increasing of proteinuria, urinary occult blood and urinary glucose was detected by urine analyzer in ß(1)-AABs group. By HE and Masson's coloration, lots of mononuclear cell infiltration and collagen fibers deposition could be observed at the 24th week of immunization. After the treatment of ß(1)-AABs, the caspase-3 activity increased significantly in the HEK293 cells transfected with ß(1)-AR-EGFP plasmids, while no significant changes were observed for lactate dehydrogenase (LDH) activity. The results indicate that long-term presence of ß(1)-AABs can induce the morphological and functional damage of the kidneys in adult and aged rats.

[The up-regulation of hypoxia inducible factor-1α by hypoxic postconditioning reduces hypoxia/reoxygenation-induced injury in heart-derived H9c2 cells].

The aim of the present study was to explore the effects of hypoxic postconditioning (PostC) on heart-derived H9c2 cells injury induced by hypoxia/reoxygenation (H/R) and the expression of hypoxia inducible factor-1α (HIF-1α), and to analyze the relationship between them. Cultured H9c2 cardiac muscle cells were subjected to 3-hour hypoxia and 2-hour reoxygenation to simulate ischemia and reperfusion, or underwent 3 cycles of 5-min reoxygenation and 5-min hypoxia preceding the long reoxygenation to simulate ischemic postconditioning. Cell viability, lactate dehydrogenase (LDH) activity, and caspase-3 activity were detected respectively to investigate the cell injury induced by H/R. The level of HIF-1α mRNA was measured by real-time PCR. Western blot was used to determine HIF-1α protein level. The results showed that postconditioning significantly increased H9c2 cell viability, reduced the activity of LDH and caspase-3. Simultaneously, postconditioning up-regulated the HIF-1α protein level. Moreover, after DMOG, an inhibitor of proline hydroxylase (PHD) which targeted to HIF-1α degradation, was used to stabilize HIF-1α protein level, the reduction of H9c2 cells injury was comparable to that by postconditioning. There was a significant linear positive relationship between HIF-1α protein level and cell viability (r = 0.743, P < 0.01). After HIF-1α gene was silenced by siRNA, the cardio-protective effects of postconditioning was significantly weakened. These data suggest that up-regulation of HIF-1α plays an important role in the cardio-protection of postconditioning.

Attenuation of myocardial injury by postconditioning: role of hypoxia inducible factor-1alpha.

Postconditioning (PostC) has regenerated interest as a mechanical intervention against myocardial ischemia/reperfusion injury, but its molecular mechanisms remain elusive. This study tested the hypothesis that hypoxia inducible factor-1alpha (HIF-1alpha) plays a role in PostC-induced cardioprotection. Male Wistar rats were subjected to 30 min ischemia followed by 3 h of reperfusion (Control). PostC with 3 cycles of 10 s reperfusion and 10 s re-occlusion was applied at the onset of reperfusion. Relative to the Sham group, HIF-1alpha protein level was increased by 2.9-fold in the Control group, but its level was enhanced by 5.8-fold with PostC (P < 0.01 vs. Control). However, HIF-1alpha protein level was further augmented by 2.0-fold and 3.3-fold, respectively, when the prolyl hydroxylase inhibitor, dimethyloxalylglycine (DMOG, 40 mg/kg, i.p.) was given at 24 h before ischemia in both Control and PostC groups. PostC reduced infarct size by 24% compared with the Control (27 +/- 4.2% vs. 36 +/- 5.2%, P < 0.01), consistent with significant lower levels of plasma creatine kinase activity, index of cardiomyocyte apoptosis and caspase-3 activity. Although pretreatment with DMOG significantly reduced infarct size relative to the Control, the infarct-sparing effect of PostC was remarkably enhanced when DMOG was given before PostC (18 +/- 2.0% vs. 27 +/- 4.2% in PostC alone, P < 0.05). There was a significant linear inverse relationship between HIF-1alpha protein level and infarct size (r = -0.799, P < 0.01) among all groups. Furthermore, along with up-regulated HIF-1alpha expression, the levels of iNOS mRNA and protein were significantly increased in the PostC alone and DMOG plus PostC groups. In conclusion, these data suggest that HIF-1alpha is involved in cardioprotection by PostC and pharmacological augmentation of HIF-1alpha expression that enhances the infarct-sparing effect of PostC; iNOS, the downstream gene of HIF-1alpha, may participate in signaling pathways in mediating PostC's protection.

A missense mutation in the CHRM2 gene is associated with familial dilated cardiomyopathy.

Circulating autoantibodies against the M2-muscarinic acetylcholine receptor (CHRM2) have been detected in patients with dilated cardiomyopathy (DCM). However, it has yet to be determined whether the pathogenesis of familial DCM may be linked to the genetic variability of the CHRM2 gene. The coding regions of the CHRM2 gene were examined by direct DNA sequencing. Plasma concentrations of autoantibodies against CHRM2 were determined by ELISA in 7 unrelated DCM families. Linkage analysis demonstrated cosegregation of the microsatellite markers, D7S509 and D7S495 that flank the CHRM2 gene, with the familial form of DCM. A novel missense mutation (C722G) replacing cysteine with tryptophane (Cys176Trp) was identified in the CHRM2 gene in all affected members but was absent in unaffected members. Additionally, 139 sporadic DCM patients and 450 normal volunteers were screened for the same mutation, but none were identified. Among the 12 affected members with familial DCM, 5 patients had died suddenly and 7 experienced ventricular arrhythmia, atrioventricular conduction block, and heart failure. All mutation carriers were positive for autoantibodies against CHRM2. Survival analysis disclosed that prognosis in patients who were mutation carriers with familial DCM was poorer than that seen in patients who were noncarriers with sporadic DCM ((P<0.05). We have identified a novel missense mutation (C722G) in the CHRM2 gene associated with familial DCM. We also show that this variant correlates with the presence of autoantibodies against CHRM2. Patients with C722G mutation have more progressive disease, characterized by sudden death, arrhythmia, and heart failure.

Hyperlipidemia does not prevent the cardioprotection by postconditioning against myocardial ischemia/reperfusion injury and the involvement of hypoxia inducible factor-1alpha upregulation.

Hyperlipidemia is regarded as an independent risk factor in the development of ischemic heart disease, and it can increase the myocardial susceptibility to ischemia/reperfusion (I/R) injury. Ischemic postconditioning (Postcon) has been demonstrated to attenuate the myocardial injury induced by I/R in normal conditions. But the effect of ischemic Postcon on hyperlipidemic animals is unknown. Hypoxia inducible factor-1 (HIF-1) has been demonstrated to play a central role in the cardioprotection by preconditioning, which is one of the protective strategies except for Postcon. The aim of this study was to determine whether Postcon could reduce myocardial injury in hyperlipidemic animals and to assess whether HIF-1 was involved in Postcon mechanisms. Male Wistar rats underwent the left anterior descending coronary occlusion for 30 min followed by 180 min of reperfusion with or without Postcon after fed with high fat diet or normal diet for 8 weeks. The detrimental indices induced by the I/R insult included infarct size, plasma creatine kinase activity and caspase-3 activity. Results showed that hyperlipidemia remarkably enhanced the myocardial injury induced by I/R, while Postcon significantly decreased the myocardial injury in both normolipidemic and hyperlipidemic rats. Moreover, both hyperlipidemia and I/R promoted the HIF-1alpha expression. Most importantly, we have for the first time demonstrated that Postcon further induced a significant increase in HIF-1alpha protein level not only in normolipidemic but also in hyperlipidemic conditions. Thus, Postcon reduces the myocardial injury induced by I/R in normal and hyperlipidemic animals, and HIF-1alpha upregulation may involve in the Postcon-mediated cardioprotective mechanisms.

Detection of serum autoantibodies against AT1A-receptor during the development of the four types of hypertensive rat models.

Using two-kidney one-clip renal hypertensive (2K1C group), stress-induced hypertensive (neural group), DOCA-salt treated hypertensive (DOCA group) and spontaneously hypertensive rats (SHR group), to investigate the change in AT(1A)-receptor autoantibodies (AT(1A)-AAs) during the development of the four types of hypertension. The biological activities of AT(1A)-AAs were examined. It was shown that the frequency of occurrence and titres of AT(1A)-AAs increased significantly during the development of hypertension. In the four hypertensive groups studied, the occurrence of AT(1A)-AAs was most prominent in SHR, 2K1C and neural groups. The biological effects of AT(1A)-AAs were shown to increase the beating frequency of cultured neonatal myocardial and vascular contractile tension. It is suggested that autoimmune mechanisms are involved the pathogenesis of different types of hypertension and the AT(1A)-AAs may be one of the mechanisms leading to cardiac hypertrophy.

[Changes in autoantibody against cardiovascular AT1-receptor during development of renovascular hypertension in rats].

The aim of this study was to observe the change in angiotensin II receptor subtype 1 (AT(1)) autoantibody during the development of renovascular hypertension (RVH). The Goldblatt renovascular hypertension model was established by the two-kidney one-clip method, and a synthetic peptide corresponding to amino acid sequence 165-191 of the second extracellular loop of the AT(1)-receptor was used as the antigen. Sera AT(1)-receptor autoantibody was detected by SA-ELISA. It was shown that two weeks after operation both the frequency of occurrence and the titre of autoantibodies to AT(1)-receptor were significantly increased as compared with the pre-treatment control. The increase in autoantibodies lasted several weeks and then decreased gradually to the pre-clipping level at 12 weeks. It is suggested that autoimmune mechanisms are involved in the pathogenesis of renovascular hypertension and the AT(1) autoantibodies may be one of the mechanisms leading to cardiac hypertrophy.

Comparative studies of vasodilating effects of angiotensin-(1-7) on the different vessels.

The purpose of this study was to compare the vasodilating effects of angiotensin-(1-7) [Ang-(1-7)] on the different vessels and to clarify its mechanisms by using relaxing responses of preconstricted vascular rings. The results showed: (1) Ang-(1-7) dose-dependently induced vasorelaxation in all the vessels studied. However, there is apparent heterogeneity in the responsiveness of vessels from different origin. (2) The Ang-(1-7)-induced vasorelaxation was endothelium dependent and largely mediated by NO system. (3) The vasodilator action of Ang-(1-7) was not mediated by AT1 or AT2 receptor subtypes. It is suggested that the Ang-(1-7)-induced vasorelaxation is endothelium dependent by some other unclarified angiotensin receptor subtypes and is largely mediated by NO system.

Autoantibodies against the ß3-adrenoceptor protect from cardiac dysfunction in a rat model of pressure overload.

ß3-Adrenoceptors (ß3-ARs) mediate a negative inotropic effect in human ventricular cardiomyocytes, which is opposite to that of ß1- and ß2-ARs. It has been previously demonstrated that autoantibodies against the ß1/ß2-AR exist in the sera of some patients with heart failure (HF) and these autoantibodies display agonist-like effects. Our aim in this study was to observe whether autoantibodies against the ß3-AR (ß3-AR Abs) exist in the sera of patients with HF and to assess the effects of ß3-AR Abs on rat model of pressure overload cardiomyopthy. In the present study, the level of ß3-AR Abs in the sera of HF patients was screened by ELISA. ß3-AR Abs from HF patients were administrated to male adult rats with abdominal aortic banding (AAB), and the cardiac function was measured by echocardiographic examination and hemodynamic studies. The biological effects of this autoantibody on cardiomyocytes were evaluated using a motion-edge detection system, intracellular calcium transient assay, and patch clamp techniques. Compared to healthy subjects, the frequency of occurrence and titer of ß3-AR Abs in the sera of HF patients were greatly increased, and ß3-AR Abs could prevent LV dilation and improve the cardiac function of rats with AAB. ß3-AR Abs exhibited negative chronotropic and inotropic effects and were accompanied by a decreased intracellular Ca(2+) transient and membrane L-type Ca(2+) current in cardiomyocytes. Our results demonstrated the existence of ß3-AR Abs in the sera of patients with HF and found that this autoantibody could alleviate the cardiac dysfunction induced by pressure-overload in AAB rats.

Proteomics screen to reveal molecular changes mediated by C722G missense mutation in CHRM2 gene.

UNLABELLED: Previously, we reported a missense mutation (C722G) in the M2-muscarinic acetylcholine receptor (CHRM2) gene associated with familial dilated cardiomyopathy. However, the exact molecular mechanisms by the related protein changes of CHRM2-C722G mutation induced are still unclear. CHRM2 and CHRM2-C722G lentiviral vector was infected to CHO cells. Proteomic analysis by label-free shotgun strategy and the STRING 9.0 software were performed. A total of 102 proteins with at least 2-fold change in the CHRM2-C722G group were identified, 42 proteins were up-regulated, whereas 57 were down-regulated. These altered proteins belong to three broad functional categories: (i) metabolic (e.g. Cytosolic acyl coenzyme A thioester hydrolase, Malate dehydrogenase); (ii) cytoskeletal (e.g. Actin-related protein, Myosin light polypeptide 6 and Alpha-actinin-1) and (iii) stress response (e.g. heat shock protein 70, Ras-related protein Rab-10). Interestingly, the marked differences in the expression of selected eight proteins (change >4.0-fold), were connected with many proteins related to apoptosis and immune/inflammatory response such as: FOS, BAX, MYC, TP53 and IL6. This novel study demonstrated for the first time a full-scale screening of the proteomics research by CHRM2-C722G mutation and profiled 102 changed proteins, of which, eight might be critical in cardiac dysfunction for future mapping. SIGNIFICANCE: It was a full-scale screening of the proteomics research by CHRM2-C722G mutation. These proteins might serve as valuable biomarkers that could predict the presence of a precursor field. These proteins might serve to further explore the pathophysiological mechanisms in familial DCM patients with C176W mutation.

[The attenuation of myocardial susceptibility to ischemia/reperfusion injury by ischemic postconditioning in hypercholesteremia rats and the role of hypoxia inducible factor-1alpha].

OBJECTIVE: To explore whether ischemic postconditioning can attenuate the myocardial injury induced by ischemia/reperfusion (I/R) in hypercholesteremic rats and whether hypoxia inducible factor-1alpha (HIF-1alpha) play a role in the protection. METHODS: Adult male Wistar rats received a high fat diet for 8 weeks to prepare the hypercholesteremic models. Myocardial damage induced by ischemia/reperfusion was evaluated by infarct size, creatine kinase (CK) activity and myocardial apoptosis. HIF-1alpha mRNA level was detected by real time-RT-PCR and the protein level was detected by Western blot. RESULTS: Myocardial infarct size, CK activity, and caspase-3 activity induced by I/R were markedly increased in hypercholesteremic rats compared with those in normal rats. Ischemic postconditioning attenuated the myocardial injury in both normal rats and hypercholesteremic rats, and increased HIF-1alpha protein level. There was a significant linear inverse relationship between HIF-1alpha protein level and infarct size (r = -0.802, P <0.01). CONCLUSION: Hypercholesteremia enhanced the susceptibility of myocardia to ischemia/reperfusion injury. While ischemic postconditioning markedly attenuated the increase of myocardial susceptibility to I/R induced by hypercholesteremia. HIF-1alpha might be one of the mechanisms of protection by ischemic postconditioning.

Long-term active immunization with a synthetic peptide corresponding to the second extracellular loop of ß1-adrenoceptor induces both morphological and functional cardiomyopathic changes in rats.

BACKGROUND: ß1-adrenoceptors (ß1-ARs) are the predominant receptors in regulating heart functions. ß1-ARs contain 7-transmembrane domains (7TM), 3 extracellular loops and 3 intracellular loops. Among these loops, the second extracellular loop of ß1-AR (ß1-AR-ECII) plays an important role in the pathogenesis of heart failure. METHODS: (1) Select the sera-negative rats for antibodies against ß1-AR-ECII. (2) Detect the level of antibodies against ß1-AR-ECII in the process of active immunization with artificial synthetic peptides according to the sequence of human ß1-AR-ECII. (3) Observe its long-term role on cardiac structure and function in vivo by immunizing rats. (4) Detect the changes of T lymphocytes subsets in the process of active immunization. RESULTS: The peptides induced the production of specific autoantibody against ß1-AR-ECII. Furthermore, immunization with ß1-AR-ECII peptide induced both morphological and functional alterations in the hearts of rats, which potentially results in heart failure. In addition, induction of the autoantibodies against ß1-AR-ECII increased the CD4+/CD8+ ratio in the peripheral blood of rats. DISCUSSION: In this study, we determined the effect of anti-ß1-AR-ECII autoantibody on morphological and functional cardiomyopathic alterations by immunization of rats with a synthetic peptide corresponding to the ß1-AR-ECII for 18 months. These results provide further evidence that autoantibody against ß1-AR-ECII is involved in the pathogenesis of heart failure. But the underlying mechanisms need further study.

Role of iNOS-derived reactive nitrogen species and resultant nitrative stress in leukocytes-induced cardiomyocyte apoptosis after myocardial ischemia/reperfusion.

Polymorphonuclear leukocyte (PMN) accumulation/activation has been implicated as a primary mechanism underlying MI/R injury. Recent studies have demonstrated that PMNs express inducible nitric oxide synthase (iNOS) and produce toxic reactive nitrogen species (RNS). However, the role of iNOS-derived reactive nitrogen species and resultant nitrative stress in PMN-induced cardiomyocyte apoptosis after MI/R remains unclear. Male adult rats were subjected to 30 min of myocardial ischemia followed by 5 h of reperfusion. Animals were randomized to receive one of the following treatments: MI/R+vehicle; MI/R+L-arginine; PMN depletion followed by MI/R+vehicle; PMN depletion followed by MI/R+L-arginine; MI/R+1400 W; MI/R+1400 W+L-arginine and MI/R+ FeTMPyP. Ischemia/reperfusion-induced and L-arginine-enhanced nitrative stress and cardiomyocyte apoptosis were determined. PMN depletion virtually abolished ischemia/reperfusion- induced PMN accumulation, attenuated ischemic/reperfusion-induced and L-arginine-enhanced nitrative stress, and reduced ischemic/reperfusion-induced and L-arginine-enhanced cardiomyocyte apoptosis (P values all <0.01). Pre-treatment with 1400 W, a highly selective iNOS inhibitor, had no effect on PMN accumulation in the ischemic/reperfused tissue. However, this treatment reduced ischemia/reperfusion-induced and L-arginine-enhanced nitrative stress and cardiomyocyte apoptosis to an extent that is comparable as that seen in PMN depletion group. Treatment with FeTMPyP, a peroxynitrite decomposition catalyst, had no effect on either PMN accumulation or total NO production. However, treatment with this ONOO(-) decomposition catalyst also reduced ischemia/reperfusion-induced and L-arginine-enhanced nitrative stress and cardiomyocyte apoptosis (P values all <0.01). These results demonstrated that ischemic/reperfusion stimulated PMN accumulation may result in cardiomyocyte injury by an iNOS-derived nitric oxide initiated and peroxynitrite-mediated mechanism. Therapeutic interventions that block PMN accumulation, inhibit iNOS activity or scavenge peroxynitrite may reduce nitrative stress and attenuate tissue injury.