MicroRNA-132 controls water homeostasis through regulating MECP2-mediated vasopressin synthesis.

Fine-tuning of the body's water balance is regulated by vasopressin (AVP), which induces the expression and apical membrane insertion of aquaporin-2 water channels and subsequent water reabsorption in the kidney. Here we demonstrate that silencing of microRNA-132 (miR-132) in mice causes severe weight loss due to acute diuresis coinciding with increased plasma osmolality, reduced renal total and plasma membrane expression of aquaporin-2, and abrogated increase in AVP levels. Infusion with synthetic AVP fully reversed the antagomir-132-induced diuresis, and low-dose intracerebroventricular administration of antagomir-132 similarly caused acute diuresis. Central and intracerebroventricular antagomir-132 injection both decreased hypothalamic AVP mRNA levels. At the molecular level, antagomir-132 increased the in vivo and in vitro mRNA expression of methyl-CpG-binding protein-2 (MECP2), which is a miR-132 target and which blocks AVP gene expression by binding its enhancer region. In line with this, treatment of hypothalamic N6 cells with a high-salt solution increased its miR-132 levels, whereas it attenuated endogenous Mecp2 mRNA levels. In conclusion, we identified miR-132 as a first miRNA regulating the osmotic balance by regulating the hypothalamic AVP gene mRNA expression.

Changes of myocardial gene expression and protein composition in patients with dilated cardiomyopathy after immunoadsorption with subsequent immunoglobulin substitution.

Immunoadsorption with subsequent immunoglobulin substitution (IA/IgG) represents a therapeutic approach for patients with dilated cardiomyopathy (DCM). Here, we studied which molecular cardiac alterations are initiated after this treatment. Transcription profiling of endomyocardial biopsies with Affymetrix whole genome arrays was performed on 33 paired samples of DCM patients collected before and 6 months after IA/IgG. Therapy-related effects on myocardial protein levels were analysed by label-free proteome profiling for a subset of 23 DCM patients. Data were analysed regarding therapy-associated differences in gene expression and protein levels by comparing responders (defined by improvement of left ventricular ejection fraction ≥20 % relative and ≥5 % absolute) and non-responders. Responders to IA/IgG showed a decrease in serum N-terminal proBNP levels in comparison with baseline which was accompanied by a decreased expression of heart failure markers, such as angiotensin converting enzyme 2 or periostin. However, despite clinical improvement even in responders, IA/IgG did not trigger general inversion of DCM-associated molecular alterations in myocardial tissue. Transcriptome profiling revealed reduced gene expression for connective tissue growth factor, fibronectin, and collagen type I in responders. In contrast, in non-responders after IA/IgG, fibrosis-associated genes and proteins showed elevated levels, whereas values were reduced or maintained in responders. Thus, improvement of LV function after IA/IgG seems to be related to a reduced gene expression of heart failure markers and pro-fibrotic molecules as well as reduced fibrosis progression.

High-density lipoproteins reduce palmitate-induced cardiomyocyte apoptosis in an AMPK-dependent manner.

Palmitate has been implicated in the induction of cardiomyocyte apoptosis via reducing the activity of 5' AMP-activated protein kinase (AMPK). We sought to evaluate whether high-density lipoproteins (HDLs), known for their cardioprotective features and their potential to increase AMPK activity, can reduce palmitate-induced cardiomyocyte apoptosis and whether this effect is AMPK-dependent. Therefore, cardiomyocytes were isolated from adult Wistar rat hearts via perfusion on a Langendorff-apparatus and cultured in free fatty acid-free BSA control medium or 0.5 mM palmitate medium in the presence or absence of HDL (5 µg protein/ml) with or without 0.1 µM of the AMPK-inhibitor compound S for the analysis of Annexin V/propidium, genes involved in apoptosis and fatty acid oxidation, and cardiomyocyte contractility. We found that HDLs decreased palmitate-induced cardiomyocyte apoptosis as indicated by a reduction in Annexin V-positive cardiomyocytes and an increase in Bcl-2 versus Bax ratio. Concomitantly, HDLs increased the palmitate-impaired expression of genes involved in fatty acid oxidation. Furthermore, HDLs improved the palmitate-impaired cardiomyocyte contractility. All effects were mediated in an AMPK-dependent manner, concluding that HDLs reduce palmitate-induced cardiomyocyte apoptosis, resulting in improved cardiomyocyte contractility through a mechanism involving AMPK.

A protein kinase A-independent pathway controlling aquaporin 2 trafficking as a possible cause for the syndrome of inappropriate antidiuresis associated with polycystic kidney disease 1 haploinsufficiency.

Renal water reabsorption is controlled by arginine vasopressin (AVP), which binds to V2 receptors, resulting in protein kinase A (PKA) activation, phosphorylation of aquaporin 2 (AQP2) at serine 256, and translocation of AQP2 to the plasma membrane. However, AVP also causes dephosphorylation of AQP2 at S261. Recent studies showed that cyclin-dependent kinases (cdks) can phosphorylate AQP2 peptides at S261 in vitro. We investigated the possible role of cdks in the phosphorylation of AQP2 and identified a new PKA-independent pathway regulating AQP2 trafficking. In ex vivo kidney slices and MDCK-AQP2 cells, R-roscovitine, a specific inhibitor of cdks, increased pS256 levels and decreased pS261 levels. The changes in AQP2 phosphorylation status were paralleled by increases in cell surface expression of AQP2 and osmotic water permeability in the absence of forskolin stimulation. R-Roscovitine did not alter cAMP-dependent PKA activity but specifically reduced protein phosphatase 2A (PP2A) expression and activity in MDCK cells. Notably, we found reduced PP2A expression and activity and reduced pS261 levels in Pkd1(+/-) mice displaying a syndrome of inappropriate antidiuresis with high levels of pS256, despite unchanged AVP and cAMP. Similar to previous findings in Pkd1(+/-) mice, R-roscovitine treatment caused a significant decrease in intracellular calcium in MDCK cells. Our data indicate that reduced activity of PP2A, secondary to reduced intracellular Ca(2+) levels, promotes AQP2 trafficking independent of the AVP-PKA axis. This pathway may be relevant for explaining pathologic states characterized by inappropriate AVP secretion and positive water balance.

Small-molecule screening identifies modulators of aquaporin-2 trafficking.

In the principal cells of the renal collecting duct, arginine vasopressin (AVP) stimulates the synthesis of cAMP, leading to signaling events that culminate in the phosphorylation of aquaporin-2 water channels and their redistribution from intracellular domains to the plasma membrane via vesicular trafficking. The molecular mechanisms that control aquaporin-2 trafficking and the consequent water reabsorption, however, are not completely understood. Here, we used a cell-based assay and automated immunofluorescence microscopy to screen 17,700 small molecules for inhibitors of the cAMP-dependent redistribution of aquaporin-2. This approach identified 17 inhibitors, including 4-acetyldiphyllin, a selective blocker of vacuolar H(+)-ATPase that increases the pH of intracellular vesicles and causes accumulation of aquaporin-2 in the Golgi compartment. Although 4-acetyldiphyllin did not inhibit forskolin-induced increases in cAMP formation and downstream activation of protein kinase A (PKA), it did prevent cAMP/PKA-dependent phosphorylation at serine 256 of aquaporin-2, which triggers the redistribution to the plasma membrane. It did not, however, prevent cAMP-induced changes to the phosphorylation status at serines 261 or 269. Last, we identified the fungicide fluconazole as an inhibitor of cAMP-mediated redistribution of aquaporin-2, but its target in this pathway remains unknown. In conclusion, our screening approach provides a method to begin dissecting molecular mechanisms underlying AVP-mediated water reabsorption, evidenced by our identification of 4-acetyldiphyllin as a modulator of aquaporin-2 trafficking.

Myocardial gene expression profiles and cardiodepressant autoantibodies predict response of patients with dilated cardiomyopathy to immunoadsorption therapy.

AIMS: Immunoadsorption with subsequent immunoglobulin G substitution (IA/IgG) represents a novel therapeutic approach in the treatment of dilated cardiomyopathy (DCM) which leads to the improvement of left ventricular ejection fraction (LVEF). However, response to this therapeutic intervention shows wide inter-individual variability. In this pilot study, we tested the value of clinical, biochemical, and molecular parameters for the prediction of the response of patients with DCM to IA/IgG. METHODS AND RESULTS: Forty DCM patients underwent endomyocardial biopsies (EMBs) before IA/IgG. In eight patients with normal LVEF (controls), EMBs were obtained for clinical reasons. Clinical parameters, negative inotropic activity (NIA) of antibodies on isolated rat cardiomyocytes, and gene expression profiles of EMBs were analysed. Dilated cardiomyopathy patients displaying improvement of LVEF (≥20 relative and ≥5% absolute) 6 months after IA/IgG were considered responders. Compared with non-responders (n = 16), responders (n = 24) displayed shorter disease duration (P = 0.006), smaller LV internal diameter in diastole (P = 0.019), and stronger NIA of antibodies. Antibodies obtained from controls were devoid of NIA. Myocardial gene expression patterns were different in responders and non-responders for genes of oxidative phosphorylation, mitochondrial dysfunction, hypertrophy, and ubiquitin-proteasome pathway. The integration of scores of NIA and expression levels of four genes allowed robust discrimination of responders from non-responders at baseline (BL) [sensitivity of 100% (95% CI 85.8-100%); specificity up to 100% (95% CI 79.4-100%); cut-off value: -0.28] and was superior to scores derived from antibodies, gene expression, or clinical parameters only. CONCLUSION: Combined assessment of NIA of antibodies and gene expression patterns of DCM patients at BL predicts response to IA/IgG therapy and may enable appropriate selection of patients who benefit from this therapeutic intervention.

Demeclocycline attenuates hyponatremia by reducing aquaporin-2 expression in the renal inner medulla.

Binding of vasopressin to its type 2 receptor in renal collecting ducts induces cAMP signaling, transcription and translocation of aquaporin (AQP)2 water channels to the plasma membrane, and water reabsorption from the prourine. Demeclocycline is currently used to treat hyponatremia in patients with the syndrome of inappropriate antidiuretic hormone secretion (SIADH). Demeclocycline's mechanism of action, which is poorly understood, is studied here. In mouse cortical collecting duct (mpkCCD) cells, which exhibit deamino-8-D-arginine vasopressin (dDAVP)-dependent expression of endogenous AQP2, demeclocycline decreased AQP2 abundance and gene transcription but not its protein stability. Demeclocycline did not affect vasopressin type 2 receptor localization but decreased dDAVP-induced cAMP generation and the abundance of adenylate cyclase 3 and 5/6. The addition of exogenous cAMP partially corrected the demeclocycline effect. As in patients, demeclocycline increased urine volume, decreased urine osmolality, and reverted hyponatremia in an SIADH rat model. AQP2 and adenylate cyclase 5/6 abundances were reduced in the inner medulla but increased in the cortex and outer medulla, in the absence of any sign of toxicity. In conclusion, our in vitro and in vivo data indicate that demeclocycline mainly attenuates hyponatremia in SIADH by reducing adenylate cyclase 5/6 expression and, consequently, cAMP generation, AQP2 gene transcription, and AQP2 abundance in the renal inner medulla, coinciding with a reduced vasopressin escape response in other collecting duct segments.

In mpkCCD cells, long-term regulation of aquaporin-2 by vasopressin occurs independent of protein kinase A and CREB but may involve Epac.

Urine concentration involves the hormone vasopressin (AVP), which stimulates cAMP production in renal principal cells, resulting in translocation and transcription of aquaporin-2 (AQP2) water channels, greatly increasing the water permeability, leading to a concentrated urine. As cAMP levels decrease shortly after AVP addition, whereas AQP2 levels still increase and are maintained for days, we investigated in the present study the mechanism responsible for the AQP2 increase after long-term 1-desamino-8-d-arginine vasopressin (dDAVP) application using mouse collecting duct (mpkCCD) cells. While 30 min of dDAVP incubation strongly increased cAMP, cAMP was lower with 1 day and was even further reduced with 4 days of dDAVP, although still significantly higher than in control cells. One day of dDAVP incubation increased AQP2 promoter-dependent transcription, which was blocked by the protein kinase A (PKA) inhibitor H89. Moreover, phosphorylation of the cAMP-responsive element binding protein (CREB) and CRE-dependent transcription was observed after short-term dDAVP stimulation. With 4 days of dDAVP, AQP2 transcription remained elevated, but this was not blocked by H89, and CRE-dependent transcription and CREB phosphorylation were not increased. Exchange factor directly activated by cAMP (Epac) 1 and 2 were found to be endogenously expressed in mpkCCD cells. Application of dDAVP increased the expression of Epac1, while Epac2 was reduced. Incubation with a specific Epac activator after dDAVP pretreatment increased both AQP2 abundance and transcription compared with cells left unstimulated the last day. In conclusion, the PKA-CRE pathway is involved in the initial rise in AQP2 levels after dDAVP stimulation but not in the long-term effect of dDAVP. Instead, long-term regulation of AQP2 may involve the activation of Epac.

Vasopressin increases S261 phosphorylation in AQP2-P262L, a mutant in recessive nephrogenic diabetes insipidus.

BACKGROUND: Mutations in the aquaporin-2 (AQP2) gene cause nephrogenic diabetes insipidus (NDI), a renal disorder characterized by polyuria due to a lacking antidiuretic response to vasopressin. While most AQP2 mutants in recessive NDI are misfolded and retained in the endoplasmic reticulum, AQP2-P262L in NDI was impaired in its vasopressin-dependent translocation from vesicles to the plasma membrane. METHODS: Vasopressin-induced translocation of AQP2 coincides with AQP2 phosphorylation at S256, S264 and T269 and dephosphorylation at S261. Since P262 lies adjacent to S261, we tested whether a changed phosphorylation could underlie AQP-P262L missorting in NDI. RESULTS: In polarized cells, AQP2-P262L expressed as a double 29/30 kDa band, whereas wt-AQP2 expressed only as a 29 kDa band. Phosphatase treatment revealed that the 30 kDa AQP2-P262L band was due to changed phosphorylation. The use of newly developed phospho-specific antibodies showed that forskolin not only increased pS256 and pT269, but, in contrast to wt-AQP2, also pS261 in AQP2-P262L. The expression of AQP2-P262L proteins in which S261 phosphorylation was prevented (S261A), however, was still missorted to vesicles/basolateral membrane, despite the absence of the 30 kDa band. CONCLUSIONS: Together, our data reveal that vasopressin induces instead of reduces the phosphorylation of S261 in AQP2-P262L, but it remains to be established whether the changed phosphorylation causes its missorting in NDI.

Regulation of AQP2 localization by S256 and S261 phosphorylation and ubiquitination.

Vasopressin-induced water reabsorption coincides with phosphorylation of aquaporin-2 (AQP2) at S256 (pS256), dephosphorylation at S261, and its translocation to the apical membrane, whereas treatment with the phorbol ester 12-tetradecanoylphorbol-13-acetate (TPA) induces AQP2 ubiquitination at K270, its internalization, and lysosomal degradation. In this study we investigated the relationship between S256 and S261 phosphorylation in AQP2 and its ubiquitination and trafficking in MDCK cells. Forskolin stimulation associated with increased pS256 and decreased pS261 AQP2, indicating that MDCK cells are a good model. After forskolin stimulation, TPA-induced ubiquitination of AQP2 preceded phosphorylation of AQP2 at S261, which in the first instance occurred predominantly on ubiquitinated AQP2. Forskolin-induced changes in pS261 were also observed for AQP2-S256A and AQP2-S256D, which constitutively localize in vesicles and the apical membrane, respectively. Although pS261 varies with forskolin as with wild-type AQP2, AQP2-S256A is not increased in its ubiquitination. Our data reveal that pS261 occurred independently of AQP2 localization and suggest that pS261 follows ubiquitination and endocytosis and may stabilize AQP2 ubiquitination and intracellular localization. The absence of increased ubiquitination of AQP2-S256A indicates that its intracellular location is due to the lack of pS256. Furthermore, AQP2-S261A and AQP2-S261D localized to vesicles, which was due to their increased ubiquitination, because changing K270 into Arg in both mutants resulted in their localization in the apical membrane. Although still increased in its ubiquitination, AQP2-S256D-S261D localized in the apical membrane. AQP2-S256D-K270R-Ub, however, localized to intracellular vesicles. Although our localization of AQP2-S261A/D is different from that of others, these data indicate that constitutive S256 phosphorylation counterbalances S261D-induced ubiquitination and internalization or changes its structure to allow distribution to the apical membrane. The vesicular localization of AQP2-S256D-K270R-Ub, however, indicates that the dominant apical sorting of S256D can again be overruled by constitutive ubiquitination. These data indicate that the membrane localization of AQP2 is determined by the balance of the extents of phosphorylation and ubiquitination.

Characterization of the human myocardial proteome in inflammatory dilated cardiomyopathy by label-free quantitative shotgun proteomics of heart biopsies.

Dilated cardiomyopathy (DCM) is characterized by contractile dysfunction leading to heart failure. The molecular changes in the human heart associated with this disease have so far mostly been addressed at the gene expression level and only a few studies have analyzed global changes in the myocardial proteome. Therefore, our objective was to investigate the changes in the proteome in patients suffering from inflammatory DCM (iDCM) and chronic viral infection by a comprehensive quantitative approach. Comparative proteomic profiling of endomyocardial biopsies (EMB) from 10 patients with iDCM (left ventricular ejection fraction <40%, symptoms of heart failure) as well as 7 controls with normal left ventricular function and histology was performed by label-free proteome analysis (LC-MS/MS). Mass spectrometric data were analyzed with the Rosetta Elucidator software package. The analysis covered a total of 485 proteins. Among the 174 proteins displaying at least a 1.3-fold change in intensity (p < 0.05), major changes were observed for mitochondrial and cytoskeletal proteins, but also metabolic pathways were affected in iDCM compared to controls. In iDCM patients, we observed decreased levels of mitochondrial proteins involved in oxidative phosphorylation and tricarboxylic acid cycle. Furthermore, deregulation of proteins of carbohydrate metabolism, the actin cytoskeleton, and extracellular matrix remodeling was observed. Proteomic observations were confirmed by gene expression data and immunohistochemistry (e.g. collagen I and VI). This study demonstrates that label-free, mass spectrometry-centered approaches can identify disease dependent alterations in the proteome from small tissue samples such as endomyocardial biopsies. Thus, this technique might allow better disease characterization and may be a valuable tool in potential clinical proteomic studies.

Effects of immunoadsorption and subsequent immunoglobulin G substitution on cardiopulmonary exercise capacity in patients with dilated cardiomyopathy.

BACKGROUND: Recent data indicate that cardiac antibodies play an active role in the pathogenesis of dilated cardiomyopathy (DCM) and may contribute to cardiac dysfunction in patients with DCM. The present study investigated the influence of immunoadsorption with subsequent immunoglobulin G substitution (IA/IgG) on cardiopulmonary exercise capacity in patients with DCM. METHODS: Sixty patients with DCM (New York Heart Association II-IV, left ventricular ejection fraction < or =45%) were included in this single-center university hospital-based case-control study. Patients either were treated with IA/IgG (n = 30) or were followed without IA/IgG (n = 30). At baseline and after 3 months, we compared echocardiographic assessment of left ventricular function and spiroergometric exercise parameters. RESULTS: In contrast to controls, left ventricular ejection fraction improved significantly in the IA/IgG group from 33.0% +/- 1.2% to 40.1% +/- 1.5% (P < .001). In the control group, spiroergometric exercise parameters did not change during follow-up. After 3 months, maximum achieved power increased in the treatment group from 114.2 +/- 7.4 to 141.9 +/- 7.9 W (P = .02). Total exercise time increased in the treatment group from 812 +/- 29 to 919 +/- 30 seconds (P < .05). Peak oxygen uptake (Vo(2)) increased from 17.3 +/- 0.9 to 21.8 +/- 1.0 mL min(-1) kg(-1) after IA/IgG (P < .01). Oxygen pulse (peak Vo(2)/maximum heart rate) increased in the treatment group (10.7 +/- 0.7 vs 13.6 +/- 0.7 mL beat(-1) min(-1), P < .01). The Vo(2) at the gas exchange anaerobic threshold increased after 3 months in the treatment group from 10.3 +/- 0.5 to 13.2 +/- 0.5 mL min(-1) kg(-1) (P < .001). The ventilatory response to exercise (V(E)/Vco(2) slope) decreased after IA/IgG therapy from 32.3 +/- 1.5 to 28.7 +/- 0.9 (P = .02). CONCLUSIONS: In patients with DCM, IA/IgG therapy may induce improvement in echocardiographic and cardiopulmonary exercise parameters.

Humoral anti-proteasomal autoimmunity in dilated cardiomyopathy.

Virus-induced chronic inflammation, autoimmune processes and impaired protein quality control may be involved in the pathogenesis of dilated cardiomyopathy (DCM). The ubiquitin-proteasome system is important in the modulation of inflammatory processes and the immune response. Proteasomes were identified as targets of a humoral autoimmune response in systemic inflammatory diseases, which provoked us to investigate anti-proteasomal immunity in DCM in detail: a total of 90 DCM patients with impaired left-ventricular function (LVEF < or = 45%) were enrolled in this study. Autoimmune response to cardiac proteasomes was found to be enhanced in DCM patients, revealing the detection of predominantly alpha subunits of the 20S proteasome complex. Proteasome antibody (ProtAb) levels were found to be particularly enhanced at stages of advanced heart failure: moderately decreased LVEF and considerably increased NT-pro BNP levels were observed in DCM patients who tested positive for ProtAb (P < 0.05). A linear regression model suggested a link between the detection of cardiotropic viruses in endomyocardial biopsies and anti-proteasomal immunity (P < 0.01). Likewise, ProtAb levels were enhanced in a murine model of chronic enterovirus myocarditis. Our data also point to a potential interaction of ProtAb with the cell surface: ProtAb exerted negative inotropic effects in field-stimulated cardiomyocytes. In conclusion, humoral autoreactive anti-proteasome immune responses appear to be enhanced in DCM. Viral infection of the myocardium may be linked to the induction of anti-proteasomal immunity in DCM.

Lack of evidence for a pathogenic role of proteasome-directed autoimmunity in dilated cardiomyopathy.

The proteasome has been identified as a target of the humoral autoimmune response in different inflammatory disease entities including dilated cardiomyopathy (DCM). However, the role of proteasome autoantibodies (ProtAb) remains to be studied. Here, we have isolated human ProtAb by affinity-purification from the IgG fractions obtained from DCM patients, which predominantly detected the outer ring subunits alpha3 of the 20S proteasome. In an attempt to study the cellular effects potentially exerted by these ProtAb, simultaneous calcium and cell contractility measurements were performed in rat cardiomyocytes revealing no short-term effects upon human ProtAb exposure. Immunofluorescence staining and FACS analysis pointed towards a failure of human ProtAb to bind to the intact cell membrane, whereas human ProtAb detected 20S proteasomes in the cytoplasm and nucleus. The lack of the cell surface interaction of human ProtAb was in agreement with the failure of these autoantibodies to interfere with the cellular viability. Further, we investigated whether the removal of ProtAb by immunoadsorption (IA) resulted in functional improvement in DCM patients. IA was performed in 90 DCM patients (left ventricular ejection fraction < or =45%, ProtAb detection at baseline in 30% of these DCM patients). Improvement of LVEF was not associated with the initial detection and removal of ProtAb in DCM patients. ProtAb were reconstituted to baseline levels as soon as after 3 months post-IA/IgG treatment despite the overall improvement of LVEF in this study group. In conclusion, our data argue against a direct impact of ProtAb in the pathogenesis of DCM.

Immunoadsorption in dilated cardiomyopathy: long-term reduction of cardiodepressant antibodies.

BACKGROUND: Disturbances of humoral immunity have been described in dilated cardiomyopathy (DCM), and a number of antibodies against cardiac cell proteins have been identified. Previous studies showed that immunoadsorption therapy with subsequent IgG substitution (IA/IgG) enhances cardiac function, and that removal of cardiodepressant antibodies may represent one essential mechanism of this therapy. The long-term effect of IA/IgG on the level of cardiodepressant antibodies remains to be elucidated. METHODS: A total of 17 patients with DCM were observed up to 12 months after IA/IgG. Echocardiographic measurements were performed at baseline, 3, 6 and 12 months after therapy. Cardiodepressant antibodies were detected by incubation of rat cardiomyocytes with purified patients' IgG and recording of contractility and Ca(2+) ratio. RESULTS: In contrast to patients without cardiodepressant antibodies before IA/IgG, patients with negative inotropic antibodies showed an improvement of left ventricular ejection fraction (LVEF) from 33.8 +/- 1.7% to 44.7 +/- 2.7%; 44.5 +/- 2.3% and 51.8 +/- 1.7% after 3, 6 and 12 months (P < 0.001 vs. baseline, P < 0.05 vs. LVEF of non-cardiodepressant group). Immediately after IA/IgG therapy, no cardiodepressant effects of patients' IgG on isolated cardiomyocytes were detectable, and this effect remained diminished until 6 months after IA/IgG (P < 0.001 for contractility and Ca(2+) ratio). Compared with the levels after 3 and 6 months, cardiodepressant antibodies reoccured after 12 months (P = 0.067 for contractility, P < 0.05 for Ca(2+) ratio vs. 6 months after IA/IgG). However, the negative inotropic reaction is still diminished compared with the reaction before IA/IgG. CONCLUSION: IA/IgG therapy induces long-term reduction of negative inotropic antibodies. After 12 months, however, re-increase of negative inotropic antibodies cannot be excluded.

Human apolipoprotein A-I gene transfer reduces the development of experimental diabetic cardiomyopathy.

BACKGROUND: The hallmarks of diabetic cardiomyopathy are cardiac oxidative stress, intramyocardial inflammation, cardiac fibrosis, and cardiac apoptosis. Given the antioxidative, antiinflammatory, and antiapoptotic potential of high-density lipoprotein (HDL), we evaluated the hypothesis that increased HDL via gene transfer (GT) with human apolipoprotein (apo) A-I, the principal apolipoprotein of HDL, may reduce the development of diabetic cardiomyopathy. METHODS AND RESULTS: Intravenous GT with 3x10(12) particles/kg of the E1E3E4-deleted vector Ad.hapoA-I, expressing human apoA-I, or Ad.Null, containing no expression cassette, was performed 5 days after streptozotocin (STZ) injection. Six weeks after apoA-I GT, HDL cholesterol levels were increased by 1.6-fold (P<0.001) compared with diabetic controls injected with the Ad.Null vector (STZ-Ad.Null). ApoA-I GT and HDL improved LV contractility in vivo and cardiomyocyte contractility ex vivo, respectively. Moreover, apoA-I GT was associated with decreased cardiac oxidative stress and reduced intramyocardial inflammation. In addition, compared with STZ-Ad.Null rats, cardiac fibrosis and glycogen accumulation were reduced by 1.7-fold and 3.1-fold, respectively (P<0.05). Caspase 3/7 activity was decreased 1.2-fold (P<0.05), and the ratio of Bcl-2 to Bax was upregulated 1.9-fold (P<0.005), translating to 2.1-fold (P<0.05) reduced total number of cardiomyocytes with apoptotic characteristics and 3.0-fold (P<0.005) reduced damaged endothelial cells compared with STZ-Ad.Null rats. HDL supplementation ex vivo reduced hyperglycemia-induced cardiomyocyte apoptosis by 3.4-fold (P<0.005). The apoA-I GT-mediated protection was associated with a 1.6-, 1.6-, and 2.4-fold induction of diabetes-downregulated phospho to Akt, endothelial nitric oxide synthase, and glycogen synthase kinase ratio, respectively (P<0.005). CONCLUSIONS: ApoA-I GT reduced the development of streptozotocin-induced diabetic cardiomyopathy.

Potential role of antibodies against cardiac Kv channel-interacting protein 2 in dilated cardiomyopathy.

BACKGROUND: Growing evidence suggests participation of autoimmune mechanisms in the pathogenesis of dilated cardiomyopathy (DCM). METHODS: Patients with heart failure (left ventricular ejection fraction < or =50%) due to DCM (n = 98) or ischemic cardiomyopathy (ICM, n = 49) and controls with normal left ventricular function (n = 98) were included. Immunoglobulin G antibodies were purified from plasma by affinity chromatography and analyzed by surface plasmon resonance analysis. We analyzed the distribution of autoantibodies against Kv channel-interacting protein (KChIP) 2.6, cardiac troponin I (cTnI), and the beta1-adrenergic receptor (second extracellular loop, cardiac beta1-adrenergic receptor [SEL-beta1-AR])-two other known autoantibodies involved in heart failure. Effects of antibodies against KChIP2 on cell death of isolated rat cardiomyocytes were assessed by flow cytometry. RESULTS: We detected autoantibodies against KChIP2.6 in 14.3% (P < .015 vs controls, P = .286 vs ICM) of the DCM samples, in 8.2% of the ICM samples (P = .304 vs controls), and in 4.1% of the control samples. Virus persistence was significantly associated with detection of autoantibodies against KChIP2.6 in DCM patients (P = .025). Antibodies against SEL-beta1-AR were more frequent in DCM samples (34.7%, P < .001 vs controls, P = .02 vs ICM) and ICM samples (16.3%, P = .083 vs control) than in control samples (7.1%). Antibodies against cTnI were more frequent in DCM samples (20.4%, P < .001 vs controls, P = .769 vs ICM) and in ICM samples (18.4%, P < .01 vs controls) than in control samples (4.1%). Antibodies against rat KChIP2 enhanced cell death in isolated rat cardiomyocytes. Immunofluorescence indicated cell surface expression of KChIP2. CONCLUSIONS: Autoantibodies against KChIP2.6, SEL-beta1-AR, and cTnI appear to be associated with DCM. Antibodies against KChIP2 may enhance cell death of rat cardiomyocytes.

Positive inotropic effects of epigallocatechin-3-gallate (EGCG) involve activation of Na+/H+ and Na+/Ca2+ exchangers.

BACKGROUND: There is evidence that the tea catechin epigallocatechin-3-gallate (EGCG) modulates myocardial contractility. However, the underlying mechanisms remain to be determined. AIMS: To study potential signalling pathways involved in EGCG-induced contractile parameters. METHODS AND RESULTS: EGCG increased fractional shortening in rat cardiac myocytes and enhanced intracellular systolic Ca2+ concentrations. In isolated rat hearts, perfusion with EGCG resulted in significant, dose-dependent increase in peak systolic left ventricular pressure, as well as in contraction and relaxation velocities. Heart rate did not change. Inhibition of the beta1-receptor with metoprolol had no influence on the contractile effects of EGCG. Furthermore, levels of cAMP and phosphorylation of phospholamban did not change with EGCG, indicating that the beta-receptor pathway is not involved. The L-type Ca2+ channel inhibitors, nifedipine and gallopamil, failed to modulate EGCG-induced increase in contractility. However, the myocardial effects and intracellular calcium transients stimulated by EGCG were significantly reduced by the antagonist of the Na+/H+ exchanger (NHE) methyl-N-isobutyl amiloride (MIA), and by blocking of the reverse mode of the Na+/Ca2+ exchanger (NCX) by KB-R7943. CONCLUSION: These results indicate that Ca2+-dependent positive inotropic and lusitropic effects of EGCG are mediated in part via activation of the Na+/H+ exchanger and the reverse mode of the Na+/Ca2+ exchanger in the rat myocardium.

Immunoadsorption and subsequent immunoglobulin substitution decreases myocardial gene expression of desmin in dilated cardiomyopathy.

Cardiac autoantibodies play a pathogenic role in dilated cardiomyopathy (DCM). Removal of antibodies by immunoadsorption (IA) induces hemodynamic improvement in DCM patients. The present study investigated the effects of IA on myocardial gene expression of the intermediate cytoskeletal filament desmin, which is upregulated in heart failure. RNA was isolated from five explanted non-failing hearts and five explanted failing hearts of DCM patients, and myocardial gene expression of desmin was estimated by real-time polymerase chain reaction (PCR). In a case-control study in six DCM patients (LVEF < 40%, NYHA II-III), IA and subsequent IgG substitution were performed at monthly intervals until month 3. Endomyocardial biopsies (EMBs) were obtained before and after IA (after 3-6 months). From six DCM patients without IA therapy (controls), EMBs were also obtained over a comparable time interval. Expression of the desmin gene was analyzed in these EMBs by real-time PCR. In failing explanted hearts, expression of desmin was significantly increased (0.88 +/- 0.12 vs 0.45 +/- 0.15 in non-failing hearts, P < 0.05). After IA, myocardial gene expression of desmin was significantly decreased (from 0.26 +/- 0.05 [baseline] to 0.14 +/- 0.04 [P < 0.05] vs baseline and controls). Removal of antibodies by IA modulates myocardial gene expression of desmin in DCM patients.