Neutralization of pathogenic beta1-receptor autoantibodies by aptamers in vivo: the first successful proof of principle in spontaneously hypertensive rats.

Autoantibodies (AABs) against the second extracellular loop of the beta1-receptor (beta1(II)-AABs) are found as a pathogenic driver in patients with idiopathic dilated cardiomyopathy, Chagas cardiomyopathy, peripartum cardiomyopathy, and myocarditis, and have been increasingly seen as a treatment target. We recently identified an aptamer (single short DNA strand) that specifically binds and neutralizes beta1(II)-AABs. Via application of this aptamer, a new treatment strategy for diseases associated with the cardio-pathogenic beta1(II)-AABs could be developed. Spontaneously hypertensive rats (SHR) positive for beta1(II)-AABs were treated five times at weekly intervals (bolus application of 2 mg/kg body weight followed by an infusion of the same amount over 20 min). SHR responded to aptamer treatment with a strong reduction in the cardio-pathogenic beta1(II)-AABs. The AABs did not substantially return within the study period. No signs for aptamer toxicity were observed by visual examination of the heart, liver, and kidney, or by measurement of plasma CK, ALT, and creatinine. The aptamer's potential for beta1(II)-AAB neutralization and consequently for cardiomyopathy treatment has been shown for the first time in vivo.

Application of mutated miR-206 target sites enables skeletal muscle-specific silencing of transgene expression of cardiotropic AAV9 vectors.

Insertion of completely complementary microRNA (miR) target sites (miRTS) into a transgene has been shown to be a valuable approach to specifically repress transgene expression in non-targeted tissues. miR-122TS have been successfully used to silence transgene expression in the liver following systemic application of cardiotropic adeno-associated virus (AAV) 9 vectors. For miR-206-mediated skeletal muscle-specific silencing of miR-206TS-bearing AAV9 vectors, however, we found this approach failed due to the expression of another member (miR-1) of the same miR family in heart tissue, the intended target. We introduced single-nucleotide substitutions into the miR-206TS and searched for those which prevented miR-1-mediated cardiac repression. Several mutated miR-206TS (m206TS), in particular m206TS-3G, were resistant to miR-1, but remained fully sensitive to miR-206. All these variants had mismatches in the seed region of the miR/m206TS duplex in common. Furthermore, we found that some m206TS, containing mismatches within the seed region or within the 3' portion of the miR-206, even enhanced the miR-206- mediated transgene repression. In vivo expression of m206TS-3G- and miR-122TS-containing transgene of systemically applied AAV9 vectors was strongly repressed in both skeletal muscle and the liver but remained high in the heart. Thus, site-directed mutagenesis of miRTS provides a new strategy to differentiate transgene de-targeting of related miRs.

Increased myocardial SERCA expression in early type 2 diabetes mellitus is insulin dependent: In vivo and in vitro data.

BACKGROUND: Calcium (Ca2+) handling proteins are known to play a pivotal role in the pathophysiology of cardiomyopathy. However little is known about early changes in the diabetic heart and the impact of insulin treatment (Ins). METHODS: Zucker Diabetic Fatty rats treated with or without insulin (ZDF ± Ins, n = 13) and lean littermates (controls, n = 7) were sacrificed at the age of 19 weeks. ZDF + Ins (n = 6) were treated with insulin for the last 6 weeks of life. Gene expression of Ca2+ ATPase in the cardiac sarcoplasmatic reticulum (SERCA2a, further abbreviated as SERCA) and phospholamban (PLB) were determined by northern blotting. Ca2+ transport of the sarcoplasmatic reticulum (SR) was assessed by oxalate-facilitated 45Ca-uptake in left ventricular homogenates. In addition, isolated neonatal cardiomyocytes were stimulated in cell culture with insulin, glucose or triiodthyronine (T3, positive control). mRNA expression of SERCA and PLB were measured by Taqman PCR. Furthermore, effects of insulin treatment on force of contraction and relaxation were evaluated by cardiomyocytes grown in a three-dimensional collagen matrix (engineered heart tissue, EHT) stimulated for 5 days by insulin. By western blot phosphorylations status of Akt was determed and the influence of wortmannin. RESULTS: SERCA levels increased in both ZDF and ZDF + Ins compared to control (control 100 ± 6.2 vs. ZDF 152 ± 26.6* vs. ZDF + Ins 212 ± 18.5*# % of control, *p < 0.05 vs. control, #p < 0.05 vs. ZDF) whereas PLB was significantly decreased in ZDF and ZDF + Ins (control 100 ± 2.8 vs. ZDF 76.3 ± 13.5* vs. ZDF + Ins 79.4 ± 12.9* % of control, *p < 0.05 vs control). The increase in the SERCA/PLB ratio in ZDF and ZDF ± Ins was accompanied by enhanced Ca2+ uptake to the SR (control 1.58 ± 0.1 vs. ZDF 1.85 ± 0.06* vs. ZDF + Ins 2.03 ± 0.1* µg/mg/min, *p < 0.05 vs. control). Interestingly, there was a significant correlation between Ca2+ uptake and SERCA2a expression. As shown by in-vitro experiments, the effect of insulin on SERCA2a mRNA expression seemed to have a direct effect on cardiomyocytes. Furthermore, long-term treatment of engineered heart tissue with insulin increased the SERCA/PLB ratio and accelerated relaxation time. Akt was significantly phosphorylated by insulin. This effect could be abolished by wortmannin. CONCLUSION: The current data demonstrate that early type 2 diabetes is associated with an increase in the SERCA/PLB ratio and that insulin directly stimulates SERCA expression and relaxation velocity. These results underline the important role of insulin and calcium handling proteins in the cardiac adaptation process of type 2 diabetes mellitus contributing to cardiac remodeling and show the important role of PI3-kinase-Akt-SERCA2a signaling cascade.

The first aptamer-apheresis column specifically for clearing blood of ß1-receptor autoantibodies.

BACKGROUND: Application of immunoapheresis to eliminate pathogenic autoantibodies targeting the second extracellular loop of the ß1-receptor (ß1-AABs) is currently investigated in patients with cardiomyopathy. Aptamers (single short DNA or RNA strands) are a new class of molecules that bind to a specific target molecule. This property qualifies aptamers for potential use in the apheresis technique. We recently identified an aptamer that specifically binds to ß1-AABs, so in the present study we tested whether this aptamer could be used as a binder to prepare an apheresis column suitable for clearing ß1-AABs from rat's blood. METHODS AND RESULTS: An apheresis column was designed containing the ß1-AAB-targeting-aptamer coupled to sepharose. As tested in vitro, this column (1) binds ß1-AABs highly specifically without marked interference with common IgGs, (2) has a capacity for clearing of approximately 1L of ß1-AAB-positive serum and (3) can be completely regenerated for subsequent use. Using the column for extracorporeal apheresis of spontaneously hypertensive rats (SHR) positive for both ß1-AABs and muscarinic 2-receptor autoantibodies (M2-AABs), only ß1-AABs were removed. In a follow-up of 9 weeks, recurrence of ß1-AABs in the blood of SHR could not be detected. CONCLUSIONS: For the first time, a newly designed apheresis column with a ß1-AAB specific aptamer as a binder was successfully used to eliminate ß1-AABs from SHR blood.

Decreased cardiac SERCA2 expression, SR Ca uptake, and contractile function in hypothyroidism are attenuated in SERCA2 overexpressing transgenic rats.

The sarco/endoplasmic reticulum (SR) Ca(2+)-ATPase SERCA2a has a key role in controlling cardiac contraction and relaxation. In hypothyroidism, decreased expression of the thyroid hormone (TH)-responsive SERCA2 gene contributes to slowed SR Ca(2+) reuptake and relaxation. We investigated whether cardiac expression of a TH-insensitive SERCA2a cDNA minigene can rescue SR Ca(2+) handling and contractile function in female SERCA2a-transgenic rats (TG) with experimental hypothyroidism. Wild-type rats (WT) and TG were rendered hypothyroid by 6-N-propyl-2-thiouracil treatment for 6 wk; control rats received no treatment. In vivo measured left ventricular (LV) hemodynamic parameters were compared with SERCA2a expression and function in LV tissue. Hypothyroidism decreased LV peak systolic pressure, dP/dt(max), and dP/dt(min) in both WT and TG. However, loss of function was less in TG. Thus slowed relaxation in hypothyroidism was found to be 1.5-fold faster in TG compared with WT (P < 0.05). In parallel, a 1.4-fold higher V(max) value of homogenate SR Ca(2+) uptake was observed in hypothyroid TG (P < 0.05 vs. hypothyroid WT), and the hypothyroidism-caused decline of LV SERCA2a mRNA expression in TG by -24% was markedly less than the decrease of -49% in WT (P < 0.05). A linear relationship was observed between the SERCA2a/PLB mRNA ratio values and the V(max) values of SR Ca(2+) uptake when the respective data of all experimental groups were plotted together (r = 0.90). The data show that expression of the TH-insensitive SERCA2a minigene compensates for loss of expressional activity of the TH-responsive native SERCA2a gene in the female hypothyroid rat heart. However, SR Ca(2+) uptake and in vivo heart function were only partially rescued.

Transgenic overexpression of heart-specific adenine nucleotide translocase 1 positively affects contractile function in cardiomyocytes.

BACKGROUND/AIMS: The adenine nucleotide translocase (ANT) exchanges ATP and ADP over the inner mitochondrial membrane, supplying the cells with energy. Interestingly, myocardial ANT1 overexpression preserves cardiac structure and function under pathophysiological conditions. To ascertain whether the contractile system is directly affected by increased ANT1 expression, we analyzed cell morphology, contraction and relaxation parameters of ANT1 transgenic (ANT1-TG) cardiomyocytes, myofibrillar protein expression, and Ca(2+) handling in ANT1-TG rat hearts. RESULTS: ANT1-TG cardiomyoycytes displayed an elevation in cell volume (52.6 ± 12.0%; p<0.0001) in comparison to wildtype (WT) cells. Concurrently, contractile function in ANT1-TG cells was significantly increased, measured by a decline in time to peak contraction (TTP) and RT50, the time from peak contraction to 50% relaxation, during stimulation with 0.5, 1, and 2 Hz. Quantification of myofibrillar proteins exhibited a marked increase in total cardiac myosin heavy chain (51.8 ± 12.8%) (p<0.03), beta myosin heavy chain (22.9 ± 5.0%; p<0.03), actin (23.8 ± 8.8%; p<0.05), and troponin I (51.5 ± 13.7%; p<0.01). Regarding intracellular Ca(2+) handling, ANT1-TGs revealed a significant elevation in sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA2a) protein level (22.2 ± 4.7%; p<0.01) associated with increased Ca(2+) uptake into the SR (34%; p<0.01). Moreover, the plasmalemmal Ca(2+) ATPase (PMCA) indicated advanced protein expression (23.8 ± 4.8%; p<0.01), whereas the protein amount of the Na(+)/Ca(2+) exchanger was not altered in ANT1 overexpressing hearts. CONCLUSION: These data reveal a close association of elevated mitochondrial ATP/ADP transportation via ANT1 with increased contractile function. Furthermore, the ANT1-TGs exhibit an elevation in SR Ca(2+) transport that contributes to increased cardiac work, which may protect the heart under pathophysiological conditions.

Genetic locus on MWF rat chromosome 6 affects kidney damage in response to L-NAME treatment in spontaneously hypertensive rats.

A major quantitative trait locus (QTL) on rat chromosome (RNO)6 was linked to albuminuria in Munich Wistar Frömter rats (MWF). We tested whether transfer of MWF RNO6 into the background of albuminuria-resistant spontaneously hypertensive rats (SHR) induces albuminuria in consomic SHR-6(MWF) animals. Male MWF, SHR, and SHR-6(MWF) were sham operated and treated between 6 and 24 wk of age with normal water (Sham) or with water containing 20 mg/l N(G)-nitro-L-arginine methyl ester (L-NAME) or unilaterally nephrectomized (Nx). Compared with SHR albuminuria was not increased in SHR-6(MWF) in both Sham and Nx groups. All animals survived the observation period in Sham and Nx groups, while premature mortality occurred from 12-14 wk on in L-NAME-treated SHR and SHR-6(MWF) compared with MWF L-NAME animals, in which survival was not affected (P < 0.005, respectively). Subsequent further analysis of L-NAME-treated animals at 12 wk of age showed significantly increased arterial blood pressures in both SHR and SHR-6(MWF) compared with control (P < 0.05), with higher levels in SHR compared with consomics (P < 0.05). However, L-NAME-treated consomic animals demonstrated increased albuminuria compared with SHR (12.7 +/- 3.5 vs. 0.8 +/- 0.2 mg/24 h; P < 0.05) and an induction of tubulointerstitial structural injury and expression of neutrophil gelatinase-associated lipocalin mRNA (P < 0.05 vs. other strains). Our study demonstrates that isolation of the RNO6 albuminuria QTL from the MWF background and transfer into SHR fails to induce an albuminuria phenotype during normal conditions or after nephron reduction. Moreover, our data indicate that genes on RNO6 contribute to the development of L-NAME-induced renal damage in the SHR strain.

Long-term cardiac-targeted RNA interference for the treatment of heart failure restores cardiac function and reduces pathological hypertrophy.

BACKGROUND: RNA interference (RNAi) has the potential to be a novel therapeutic strategy in diverse areas of medicine. Here, we report on targeted RNAi for the treatment of heart failure, an important disorder in humans that results from multiple causes. Successful treatment of heart failure is demonstrated in a rat model of transaortic banding by RNAi targeting of phospholamban, a key regulator of cardiac Ca(2+) homeostasis. Whereas gene therapy rests on recombinant protein expression as its basic principle, RNAi therapy uses regulatory RNAs to achieve its effect. METHODS AND RESULTS: We describe structural requirements to obtain high RNAi activity from adenoviral and adeno-associated virus (AAV9) vectors and show that an adenoviral short hairpin RNA vector (AdV-shRNA) silenced phospholamban in cardiomyocytes (primary neonatal rat cardiomyocytes) and improved hemodynamics in heart-failure rats 1 month after aortic root injection. For simplified long-term therapy, we developed a dimeric cardiotropic adeno-associated virus vector (rAAV9-shPLB) to deliver RNAi activity to the heart via intravenous injection. Cardiac phospholamban protein was reduced to 25%, and suppression of sacroplasmic reticulum Ca(2+) ATPase in the HF groups was rescued. In contrast to traditional vectors, rAAV9 showed high affinity for myocardium but low affinity for liver and other organs. rAAV9-shPLB therapy restored diastolic (left ventricular end-diastolic pressure, dp/dt(min), and tau) and systolic (fractional shortening) functional parameters to normal ranges. The massive cardiac dilation was normalized, and cardiac hypertrophy, cardiomyocyte diameter, and cardiac fibrosis were reduced significantly. Importantly, no evidence was found of microRNA deregulation or hepatotoxicity during these RNAi therapies. CONCLUSIONS: Our data show for the first time the high efficacy of an RNAi therapeutic strategy in a cardiac disease.

Cardiac-targeted RNA interference mediated by an AAV9 vector improves cardiac function in coxsackievirus B3 cardiomyopathy.

RNA interference (RNAi) has potential to be a novel therapeutic strategy in diverse areas of medicine. In this paper, we report on targeted RNAi for the treatment of a viral cardiomyopathy, which is a major cause of sudden cardiac death or terminal heart failure in children and young adults. RNAi therapy employs small regulatory RNAs to achieve its effect, but in vivo use of synthetic small interfering RNAs is limited by instability in plasma and low transfer into target cells. We instead evaluated an RNAi strategy using short hairpin RNA (shRdRp) directed at the RNA polymerase (RdRP) of coxsackievirus B3 (CoxB3) in HeLa cells, primary rat cardiomyocytes (PNCMs) and CoxB3-infected mice in vivo. A conventional AAV2 vector expressing shRdRp protected HeLa against virus-induced death, but this vector type was unable to transduce PNCMs. In contrast, an analogous pseudotyped AAV2.6 vector was protective also in PNCMs and reduced virus replication by >3 log10 steps. Finally, we evaluated the intravenous treatment of mice with an AAV2.9-shRdRp vector because AAV9 carries the most cardiotropic AAV capsid currently known for in vivo use. Mice with CoxB3 cardiomyopathy had disturbed left ventricular (LV) function with impaired parameters of contractility (dP/dtmax = 3,006 +/- 287 vs. 7,482 +/- 487 mmHg/s, p < 0.01) and diastolic relaxation (dP/dtmin = -2,224 +/- 195 vs. -6,456 +/- 356 mmHg/s, p < 0.01 and Tau = 16.2 +/- 1.1 vs. 10.7 +/- 0.6 ms, p < 0.01) compared to control mice. AAV2.9-shRdRp treatment significantly attenuated the cardiac dysfunction compared to control vector-treated mice on day 10 after CoxB3 infection: dP/dtmax = 3,865 +/- 354 vs. 3,006 +/- 287 mmHg/s (p < 0.05), dP/dtmin = -3,245 +/- 231 vs. -2,224 +/- 195 mmHg/s (p < 0.05) and Tau = 11.9 +/- 0.5 vs. 16.2 +/- 1.1 ms (p < 0.01). The data show, for the first time, that intravenously injected AAV9 has the potential to target RNAi to the heart and suggest AAV9-shRNA vectors as a novel therapeutic approach for cardiac disorders.

Protective effect of female gender on the development of albuminuria in a polygenetic rat model is enhanced further by replacement of a major autosomal QTL.

Clinical and experimental studies indicate that the progression of renal disease is faster in males than females. These observations are corroborated by a sexual dimorphism observed in the polygenetic MWF (Munich Wistar Frömter) rat model. The age-dependent spontaneous progression of increased UAE (urinary albumin excretion) in male MWF rats is influenced by multiple QTLs (quantitative trait loci). In contrast, female MWF rats only develop a slight increase in UAE, while the role of genetic factors for this phenotype is unknown. In the present study, we show that, compared with resistant SHRs (spontaneously hypertensive rats), both male and female MWF rats develop a significant increase in UAE at 24 weeks of age (P<0.0001), although blood pressures were lower compared with SHRs (P<0.0001). UAE was significantly higher in male (7-fold) compared with female MWF rats (162.6+/-15.9 compared with 24.0+/-5.5 mg/24 h respectively; P<0.0001), and only male MWF rats developed significant glomerulosclerosis and tubulointerstitial damage in the kidney (P<0.0001). To test the role of genetic factors in the development of low grade albuminuria in female MWF rats, we analysed the role of a major UAE QTL on rat chromosome 6. To this end, we analysed a consomic MWF-6(SHR) strain in which chromosome 6 from SHRs was introgressed into the MWF rat background. Time course analysis of UAE in females indicated that the small increase in UAE in MWF rats was fully suppressed by exchange of rat chromosome 6. Thus, taken together with previous studies in males, we show that RNO6 protects against the increase in albuminuria with age in both female and male MWF rats.

Soluble vascular endothelial growth factor receptor-1 (sFLT-1) mediates downregulation of FLT-1 and prevents activated neutrophils from women with preeclampsia from additional migration by VEGF.

Neutrophil activation and increased migration is associated with preeclampsia and is resolved after delivery. Preeclampsia is an inflammatory disorder where altered levels of vascular endothelial growth factor (VEGF) and the circulating soluble fms-like tyrosine kinase 1 (sFlt-1) have a pathogenic role. VEGF, by binding to FLT-1, induces leukocytic chemotaxis. We studied expression and function of FLT-1 in maternal neutrophils during preeclampsia and normal pregnancies. Analysis of maternal neutrophils showed the relationship between FLT-1 expression and week of gestation. Preeclamptic women express lower FLT-1 and sFLT-1 in neutrophils. In contrast, serum levels of sFLT-1 in patients with preeclampsia are increased and, therefore, inhibit upregulation of FLT-1 in neutrophils by neutralizing VEGF. VEGF-dependent FLT-1 expression is regulated by changing FLT-1-promoter activity. Promoter activity is decreased by sFLT-1. In vitro experiments demonstrated that migration of neutrophils is regulated by VEGF via FLT-1 and excess of sFLT-1. Thus, VEGF-dependent migration of neutrophils is decreased during preeclampsia as a consequence of excess circulating sFlt1. But, they still increase migration by fMLP and, therefore, migration of neutrophils from preeclamptic women is highly activated when compared with the normotensive group. In conclusion, besides being involved in inducing an antiangiogenic state in the serum, excess of sFLT-1 seems to prevent activated neutrophils from women with preeclampsia from additional migration by VEGF. We provide evidence that neutrophils may be involved in the pathophysiology of pregnancy-related hypertensive disorders.

Silencing Genes in the Heart.

Silencing of cardiac genes by RNA interference (RNAi) has developed into a powerful new method to treat cardiac diseases. Small interfering (si)RNAs are the inducers of RNAi, but cultured primary cardiomyocytes and heart are highly resistant to siRNA transfection. This can be overcome by delivery of small hairpin (sh)RNAs or artificial microRNA (amiRNAs) by cardiotropic adeno-associated virus (AAV) vectors. Here we describe as example of the silencing of a cardiac gene, the generation and cloning of shRNA, and amiRNAs directed against the cardiac protein phospholamban. We further describe the generation of AAV shuttle plasmids with self complementary vector genomes, the production of AAV vectors in roller bottles, and their purification via iodixanol gradient centrifugation and concentration with filter systems. Finally we describe the preparation of primary neonatal rat cardiomyocytes (PNRC), the transduction of PNRC with AAV vectors, and the maintenance of the transduced cell culture.

Aptamer BC007 for neutralization of pathogenic autoantibodies directed against G-protein coupled receptors: A vision of future treatment of patients with cardiomyopathies and positivity for those autoantibodies.

Cardiomyopathies such as idiopathic dilated cardiomyopathy (DCM), Chagas' cardiomyopathy and Peripartum cardiomyopathy present with autoantibodies against G-protein coupled receptors (GPCR-AABs) that agonistically activate their receptors. For the treatment of "agonistic autoantibody diseases" and in particular DCM, the removal of the GPCR-AABs by immunoadsorption (IA) has been studied with convincing patient benefit. To overcome cost and logistics problems of IA, the application of the aptamer BC007 for in vivo neutralization of GPCR-AABs could help. We demonstrate here, that the aptamer neutralized, in vitro, the presently known cardiovascular-pathogenic GPCR-AABs. In spontaneously hypertensive rats, the aptamer demonstrated its GPCR-AAB neutralizing potency in vivo. In the serum of DCM patients, the same GPCR-AAB reduction was achieved when patients were either immunoadsorbed or patient's serum was ex vivo treated with the aptamer. In our view, aptamer BC007 treatment in GPCR-AAB-positive patients would have a comparable benefit as that seen after IA. Not knowing all that interfering with our idea of aptamer-dependent neutralization of GPCR-AABs, the first preliminary steps have been taken for bringing the idea closer to patients.

Endothelin-A receptor blockade prevents left ventricular hypertrophy and dysfunction in salt-sensitive experimental hypertension.

BACKGROUND: Salt-sensitive hypertension represents a major cause of left ventricular (LV) dysfunction. We therefore explored the potential effects of the selective endothelin-A (ETA) receptor antagonist darusentan on the development of hypertension, LV hypertrophy (LVH), and dysfunction in a genetic rat model of salt-sensitive hypertension. METHODS AND RESULTS: Animals from the salt-sensitive Sabra rat strain (SBH/y) and the salt-resistant strain (SBN/y) were treated with either normal diet (SBH/y and SBN/y) or with deoxycorticosterone-acetate (DOCA) and salt (SBN/y-DOCA and SBH/y-DOCA). Additional groups were treated with 50 mg x kg(-1) x d(-1) of darusentan (SBH/y-DOCA-DA and SBN/y-DOCA-DA). Systolic blood pressure and LV weight increased in response to DOCA only in the SBH/y strain (+75 mm Hg and +30%; P<0.05). LV end-diastolic pressure increased and -dP/dtmax decreased in SBH/y-DOCA compared with SBH/y (P<0.05). This was paralleled by a 5-fold upregulation of LV mRNA expression of atrial natriuretic factor (ANF) and a significant reduction of sarcoplasmic reticulum (SR) Ca2+-reuptake and the SR Ca2+-ATPase to phospholamban protein ratio (-30%). Whereas treatment with darusentan in SBH/y-DOCA-DA reduced the SBP increase by 50%, LVH elevation of ANF mRNA and LV dysfunction were completely prevented (P<0.05); this was associated with a normalization of SR Ca2+-reuptake and SR Ca2+-ATPase to phospholamban ratio by darusentan (P<0.05). A moderate elevation of interstitial fibrosis in SBH/y-DOCA (P<0.05) remained unaffected by darusentan treatment. CONCLUSION: In the Sabra model of salt-sensitive hypertension, ETA-receptor blockade demonstrated striking effects on the prevention of LVH and LV dysfunction beyond its considerable antihypertensive effect.

Induction of coxsackievirus-adenovirus-receptor expression during myocardial tissue formation and remodeling: identification of a cell-to-cell contact-dependent regulatory mechanism.

BACKGROUND: The coxsackievirus-adenovirus receptor (CAR) was cloned as a receptor for both viruses, but its primary biological functions and regulatory mechanisms are unknown. CAR was low in healthy adult myocardium, whereas strong CAR reexpression was observed in human dilated cardiomyopathy. The molecular mechanisms of CAR induction in cardiomyocytes are unknown. METHODS AND RESULTS: We report on CAR regulation during development, CAR induction after myocardial infarction, and cell-to-cell contact-dependent CAR regulation in the rat. The high CAR expression during development in various organs decreased up to 190-fold after birth. After infarction resulting in severe cardiac dysfunction (dP/dt(max), -53%; dP/dt(min), -58%; left ventricular pressure, -45%), CAR was induced locally in cardiomyocytes of the infarct zone, where it was also expressed by capillary-like CD31+ structures and CD18+ interstitial cells, whereas it remained confined to subendothelial layers of arterioles and venules. In cultured cardiomyocytes, endothelin-1, cardiotrophin-1, leukemia-inhibiting factor, and cyclic stretch had no effect on CAR, whereas at high versus low cell density, CAR was suppressed up to 10-fold (P=0.006). Conditioned media from low- or high-density cardiomyocytes or cardiofibroblasts had no effect. CONCLUSIONS: The locally confined CAR upregulation after infarction makes induction by various humoral factors unlikely, because cardiac dysfunction results in high activities of sympathetic and renin-angiotensin systems and cytokines. The cell culture experiments identify a cell-to-cell contact-dependent mechanism of CAR regulation. Further characterization of the signals linking cell-to-cell interactions to CAR gene expression may provide insight into mechanisms and functional consequences of the generalized CAR induction in dilated cardiomyopathy, and of its local induction after myocardial infarction.

Sustained increase of extracellular calcium concentration causes arterial vasoconstriction in humans.

BACKGROUND: Vascular tone is affected by extracellular electrolytes. OBJECTIVE: To evaluate whether a sustained increase in the extracellular calcium concentration may produce vasoconstriction in humans. DESIGN: Cross-sectional data of a cohort study of 65 patients with end-stage renal failure. MEASUREMENTS: Arterial tone was obtained from radial artery waveforms. Intracellular stored calcium and sarcoendoplasmic reticulum Ca(2+)-ATPase activity were measured in mononuclear leukocytes using fluorescent dye techniques and oxalate-supported calcium uptake. RESULTS: During the haemodialysis sessions the extracellular calcium concentration increased significantly from 2.28 +/- 0.03 to 2.63 +/- 0.03 mmol/l (n = 65; mean +/- SEM; P < 0.001) and arterial tone increased from 31 +/- 2 to 44 +/- 3 mmHg/ml (n = 65; P < 0.001). Multivariate analysis showed that pre and postdialysis extracellular calcium and pre and postdialysis body weight were the only independent predictors of arterial tone during haemodialysis. Intracellular stored calcium in mononuclear leukocytes significantly declined from 5.1 +/- 1.2 arbitrary units at the start to 2.3 +/- 0.6 arbitrary units at the end of haemodialysis (n = 10; P = 0.01). The activity of the sarcoendoplasmic reticulum Ca(2+)-ATPase significantly decreased from 13.6 +/- 2.6 nmol calcium/mg protein per 5 min at the start to 9.2 +/- 1.6 nmol calcium/mg protein per 5 min at the end of haemodialysis (n = 28; P = 0.01). On the other hand, when a low dialysate calcium concentration was used, the increase in artery tone and reduction in sarcoendoplasmic reticulum Ca(2+)-ATPase activity were reversed. CONCLUSION: A sustained increase in the extracellular calcium concentration causes arterial vasoconstriction in humans. In addition, a reduction of sarcoendoplasmic reticulum Ca(2+)-ATPase activity and intracellular stored calcium in mononuclear leukocytes was observed.

Improvement of defective sarcoplasmic reticulum Ca2+ transport in diabetic heart of transgenic rats expressing the human kallikrein-1 gene.

The bradykinin-forming enzyme kallikrein-1 is expressed in the heart. To examine whether contractile performance and sarcoplasmic reticulum Ca2+ transport of the diabetic heart can be rescued by targeting the kallikrein-kinin system, we studied left ventricular function and sarcoplasmic reticular Ca2+ uptake after induction of streptozotocin-induced diabetes mellitus in transgenic rats expressing the human tissue kallikrein-1 gene. Six weeks after a single injection of either streptozotocin (70 mg/kg ip) or vehicle, left ventricular performance was determined using a Millar-Tip catheter system. The Ca2+-transporting activity of reticulum-derived membrane vesicles was determined in left ventricular homogenates as oxalate-supported 45Ca2+ uptake. Western blot analysis was used to quantify the reticular Ca2+-ATPase SERCA2a, phospholamban, and the phosphorylation status of the latter. Contractile performance and Ca2+ uptake activity were similar in nondiabetic wild-type and transgenic rats. Severely diabetic wild-type animals exhibited impaired left ventricular performance and decreased reticular Ca2+ uptake (-39% vs. wild-type rats, P<0.05, respectively). These changes were attenuated in diabetic transgenic rats that, in addition, exhibited a markedly increased phospholamban phosphorylation at the Ca2+/calmodulin kinase-specific site threonine17 (2.2-fold vs. diabetic wild-type rats, P<0.05). These transgene-related effects were abolished after treatment with the bradykinin B2 receptor antagonist icatibant (Hoe 140). The SERCA2-to-phospholamban ratio, phosphoserine16-phospholamban levels, and the apparent affinity for Ca2+ of the uptake reaction did not differ between the groups. Increasing the activity of the kallikrein-kinin system by expressing a human kallikrein-1 transgene protects rat heart against diabetes-induced contractile and reticular Ca2+ transport dysfunctions. An increased phosphorylation of the SERCA2 regulatory protein phospholamban at threonine17 via a B2 receptor-mediated mechanism is thereby involved.

Simulated microgravity alters differentiation and increases apoptosis in human follicular thyroid carcinoma cells.

This study focuses on the effects of simulated microgravity (0g) on the human follicular thyroid carcinoma cell line ML-1. Cultured on a three-dimensional clinostat, ML-1 cells formed three-dimensional MCTSs (MCTS diameter: 0.3 +/- 0.01 mm). After 24 and 48 h of clinorotation, the cells significantly decreased fT3 and fT4 secretion but up-regulated the thyroid-stimulating hormone-receptor expression as well as the production of vimentin, vinculin, and extracellular matrix proteins (collagen I and III, laminin, fibronectin, chondroitin sulfate) compared with controls. Furthermore, ML-1 cells grown on the clinostat showed elevated amounts of the apoptosis-associated Fas protein, of p53, and of bax but showed reduced quantities of bcl-2. In addition, signs of apoptosis became detectable, as assessed by terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick end labeling, 4', 6-diamidino-2-phenylindole staining, DNA laddering, and 85-kDa apoptosis-related cleavage fragments. These fragments resulted from enhanced 116-kDa poly(ADP-ribose)polymerase (PARP) activity and apoptosis. These observations suggest that clinorotation elevates intermediate filaments, cell adhesion molecules, and extracellular matrix proteins and simultaneously induces apoptosis in follicular thyroid cancer cells. In conclusion, our experiments could provide a regulatory basis for the finding that astronauts show low thyroid hormone levels after space flight, which may be explained by the increase of apoptosis in thyrocytes as a result of simulated 0g.

Transgenic overexpression of the sarcoplasmic reticulum Ca2+ATPase improves reticular Ca2+ handling in normal and diabetic rat hearts.

Slowed relaxation in diabetic cardiomyopathy (CM) is partially related to diminished expression of the sarcoplasmic reticulum (SR) Ca2+-ATPase SERCA2a. To evaluate the impact of SERCA2a overexpression on SR Ca2+ handling in diabetic CM, we 1) generated transgenic rats harboring a human cytomegalovirus enhancer/chicken beta-actin promotor-controlled rat SERCA2 transgene (SERCA2-TGR), 2) characterized their SR phenotype, and 3) examined whether transgene expression may rescue SR Ca2+ transport in streptozotocin-induced diabetes. The transgene was expressed in all heart chambers. Compared to wild-type (WT) rats, a heterozygous line exhibited increased SERCA2 mRNA (1.5-fold), SERCA2 protein (+26%) and SR Ca2+ uptake (+37%). Phospholamban expression was not altered. In SERCA2-TGR, contraction amplitude (+48%) and rates of contraction (+34%) and relaxation (+35%) of isolated papillary muscles (PM) were increased (P2+ uptake and SERCA2 protein of SERCA2-TGR were 1.3-fold higher (P2+ uptake, accelerates relaxation and compensates, in part, for depressed Ca2+ uptake in diabetic CM. Therefore, SERCA2 expression might constitute an important therapeutic target to rescue cardiac SR Ca2+ handling in diabetes.

A novel artificial microRNA expressing AAV vector for phospholamban silencing in cardiomyocytes improves Ca2+ uptake into the sarcoplasmic reticulum.

In failing rat hearts, post-transcriptonal inhibition of phospholamban (PLB) expression by AAV9 vector-mediated cardiac delivery of short hairpin RNAs directed against PLB (shPLBr) improves both impaired SERCA2a controlled Ca2+ cycling and contractile dysfunction. Cardiac delivery of shPLB, however, was reported to cause cardiac toxicity in canines. Thus we developed a new AAV vector, scAAV6-amiR155-PLBr, expressing a novel engineered artificial microRNA (amiR155-PLBr) directed against PLB under control of a heart-specific hybrid promoter. Its PLB silencing efficiency and safety were compared with those of an AAV vector expressing shPLBr (scAAV6-shPLBr) from an ubiquitously active U6 promoter. Investigations were carried out in cultured neonatal rat cardiomyocytes (CM) over a period of 14 days. Compared to shPLBr, amiR155-PLBr was expressed at a significantly lower level, resulting in delayed and less pronounced PLB silencing. Despite decreased knockdown efficiency of scAAV6-amiR155-PLBr, a similar increase of the SERCA2a-catalyzed Ca2+ uptake into sarcoplasmic reticulum (SR) vesicles was observed for both the shPLBr and amiR155-PLBr vectors. Proteomic analysis confirmed PLB silencing of both therapeutic vectors and revealed that shPLBr, but not the amiR155-PLBr vector, increased the proinflammatory proteins STAT3, STAT1 and activated STAT1 phosphorylation at the key amino acid residue Tyr701. Quantitative RT-PCR analysis detected alterations in the expression of several cardiac microRNAs after treatment of CM with scAAV6-shPLBr and scAAV6-amiR155-PLBr, as well as after treatment with its related amiR155- and shRNAs-expressing control AAV vectors. The results demonstrate that scAAV6-amiR155-PLBr is capable of enhancing the Ca2+ transport function of the cardiac SR PLB/SERCA2a system as efficiently as scAAV6-shPLBr while offering a superior safety profile.