Effect of Cardiotonic Steroid Marinobufagenin on Vascular Remodeling and Cognitive Impairment in Young Dahl-S Rats.

The hypertensive response in Dahl salt-sensitive (DSS) rats on a high-salt (HS) diet is accompanied by central arterial stiffening (CAS), a risk factor for dementia, and heightened levels of a prohypertensive and profibrotic factor, the endogenous Na/K-ATPase inhibitor marinobufagenin (MBG). We studied the effect of the in vivo administration of MBG or HS diet on blood pressure (BP), CAS, and behavioral function in young DSS rats and normotensive Sprague-Dawley rats (SD), the genetic background for DSS rats. Eight-week-old male SD and DSS rats were given an HS diet (8% NaCl, n = 18/group) or a low-salt diet (LS; 0.1% NaCl, n = 14-18/group) for 8 weeks or MBG (50 µg/kg/day, n = 15-18/group) administered via osmotic minipumps for 4 weeks in the presence of the LS diet. The MBG-treated groups received the LS diet. The systolic BP (SBP); the aortic pulse wave velocity (aPWV), a marker of CAS; MBG levels; spatial memory, measured by a water maze task; and tissue collection for the histochemical analysis were assessed at the end of the experiment. DSS-LS rats had higher SBP, higher aPWV, and poorer spatial memory than SD-LS rats. The administration of stressors HS and MBG increased aPWV, SBP, and aortic wall collagen abundance in both strains vs. their LS controls. In SD rats, HS or MBG administration did not affect heart parameters, as assessed by ECHO vs. the SD-LS control. In DSS rats, impaired whole-heart structure and function were observed after HS diet administration in DSS-HS vs. DSS-LS rats. MBG treatment did not affect the ECHO parameters in DSS-MBG vs. DSS-LS rats. The HS diet led to an increase in endogenous plasma and urine MBG levels in both SD and DSS groups. Thus, the prohypertensive and profibrotic effect of HS diet might be partially attributed to an increase in MBG. The prohypertensive and profibrotic functions of MBG were pronounced in both DSS and SD rats, although quantitative PCR revealed that different profiles of profibrotic genes in DSS and SD rats was activated after MBG or HS administration. Spatial memory was not affected by HS diet or MBG treatment in either SD or DSS rats. Impaired cognitive function was associated with higher BP, CAS, and cardiovascular remodeling in young DSS-LS rats, as compared to young SD-LS rats. MBG and HS had similar effects on the cardiovascular system and its function in DSS and SD rats, although the rate of change in SD rats was lower than in DSS rats. The absence of a cumulative effect of increased aPWV and BP on spatial memory can be explained by the cerebrovascular and brain plasticity in young rats, which help the animals to tolerate CAS elevated by HS and MBG and to counterbalance the profibrotic effect of heightened MBG.

Tropomyosin pseudo-phosphorylation results in dilated cardiomyopathy.

Phosphorylation of cardiac sarcomeric proteins plays a major role in the regulation of the physiological performance of the heart. Phosphorylation of thin filament proteins, such as troponin I and T, dramatically affects calcium sensitivity of the myofiber and systolic and diastolic functions. Phosphorylation of the regulatory protein tropomyosin (Tpm) results in altered biochemical properties of contraction; however, little is known about the physiological effect of Tpm phosphorylation on cardiac function. To address the in vivo significance of Tpm phosphorylation, here we generated transgenic mouse lines having a phosphomimetic substitution in the phosphorylation site of α-Tpm (S283D). High expression of Tpm S283D variant in one transgenic mouse line resulted in an increased heart:body weight ratio, coupled with a severe dilated cardiomyopathic phenotype resulting in death within 1 month of birth. Moderate Tpm S283D mice expression in other lines caused mild myocyte hypertrophy and fibrosis, did not affect lifespan, and was coupled with decreased expression of extracellular signal-regulated kinase 1/2 kinase signaling. Physiological analysis revealed that the transgenic mice exhibit impaired diastolic function, without changes in systolic performance. Surprisingly, we observed no alterations in calcium sensitivity of the myofibers, cooperativity, or calcium-ATPase activity in the myofibers. Our experiments also disclosed that casein kinase 2 plays an integral role in Tpm phosphorylation. In summary, increased expression of pseudo-phosphorylated Tpm impairs diastolic function in the intact heart, without altering calcium sensitivity or cooperativity of myofibers. Our findings provide the first extensive in vivo assessment of Tpm phosphorylation in the heart and its functional role in cardiac performance.

Dietary Sodium Restriction Reduces Arterial Stiffness, Vascular TGF-ß-Dependent Fibrosis and Marinobufagenin in Young Normotensive Rats.

High salt (HS) intake stimulates the production of marinobufagenin (MBG), an endogenous steroidal Na/K-ATPase ligand, which activates profibrotic signaling. HS is accompanied by a blood pressure (BP) increase in salt-sensitive hypertension, but not in normotensive animals. Here, we investigated whether HS stimulates MBG production and activates transforming growth factor-beta (TGF-ß) profibrotic signaling in young normotensive rats, and whether these changes can be reversed by reducing salt to a normal salt (NS) level. Three-month old male Sprague⁻Dawley rats received NS for 4 and 8 weeks (0.5% NaCl; NS4 and NS8), or HS for 4 and 8 weeks (4% NaCl; HS4 and HS8), or HS for 4 weeks followed by NS for 4 weeks (HS4/NS4), n = 8/group. Systolic BP (SBP), pulse wave velocity (PWV), MBG excretion, aortic collagen 1α2, collagen 4α1 and TGF-ß, Smad2, Smad3, Fli-1 mRNA, and total collagen abundance were measured at baseline (BL), and on weeks 4 and 8. Statistical analysis was performed using one-way ANOVA. SBP was not affected by HS (125 ± 5 and 126 ± 6 vs. 128 ± 7 mmHg, HS4 and HS8 vs. BL, p > 0.05). HS increased MBG (164 ± 19 vs. 103 ± 19 pmol/24 h/kg, HS4 vs. BL, p < 0.05) and PWV (3.7 ± 0.2 vs. 2.7 ± 0.2 m/s, HS4 vs. NS4, p < 0.05). HS8 was associated with a further increase in MBG and PWV, with an increase in aortic Col1a2 80%), Col4a1 (50%), Tgfb1 (30%), Smad2 (30%) and Smad3 (45%) mRNAs, and aortic wall collagen (180%) vs. NS8 (all p < 0.05). NS following HS downregulated HS-induced factors: in HS4/NS4, the MBG level was 91 ± 12 pmol/24 h/kg (twofold lower than HS8, p < 0.01), PWV was 3.7 ± 0.3 vs. 4.7 ± 0.2 m/s (HS4/NS4 vs. HS8, p < 0.05), aortic wall Tgfb1, Col1a2, Col4a1, Smad2, Smad3 mRNAs, and collagen abundance were reversed by salt reduction to the BL levels (p < 0.05). HS was associated with an activation of TGF-ß signaling, aortic fibrosis and aortic stiffness accompanied by an MBG increase in the absence of SBP changes in young normotensive rats. The reduction of dietary salt following HS decreased MBG, PWV, aortic wall collagen and TGF-ß. Thus, HS-induced aortic stiffness in normotensive animals occurred in the context of elevated MBG, which may activate SMAD-dependent TGF-ß pro-fibrotic signaling. This data suggests that a decrease in salt consumption could help to restore aortic elasticity and diminish the risk of cardiovascular disease by reducing the production of the pro-fibrotic factor MBG.

Cardiac remodeling in the mouse model of Marfan syndrome develops into two distinctive phenotypes.

Marfan syndrome (MFS) is a systemic disorder of connective tissue caused by mutations in fibrillin-1. Cardiac dysfunction in MFS has not been characterized halting the development of therapies of cardiac complication in MFS. We aimed to study the age-dependent cardiac remodeling in the mouse model of MFS FbnC1039G+/- mouse [Marfan heterozygous (HT) mouse] and its association with valvular regurgitation. Marfan HT mice of 2-4 mo demonstrated a mild hypertrophic cardiac remodeling with predominant decline of diastolic function and increased transforming growth factor-ß canonical (p-SMAD2/3) and noncanonical (p-ERK1/2 and p-p38 MAPK) signaling and upregulation of hypertrophic markers natriuretic peptides atrium natriuretic peptide and brain natriuretic peptide. Among older HT mice (6-14 mo), cardiac remodeling was associated with two distinct phenotypes, manifesting either dilated or constricted left ventricular chamber. Dilatation of left ventricular chamber was accompanied by biochemical evidence of greater mechanical stress, including elevated ERK1/2 and p38 MAPK phosphorylation and higher brain natriuretic peptide expression. The aortic valve regurgitation was registered in 20% of the constricted group and 60% of the dilated group, whereas mitral insufficiency was observed in 40% of the constricted group and 100% of the dilated group. Cardiac dysfunction was not associated with the increase of interstitial fibrosis and nonmyocyte proliferation. In the mouse model fibrillin-1, haploinsufficiency results in the early onset of nonfibrotic hypertrophic cardiac remodeling and dysfunction, independently from valvular abnormalities. MFS heart is vulnerable to stress-induced cardiac dilatation in the face of valvular regurgitation, and stress-activated MAPK signals represent a potential target for cardiac management in MFS.

Gi-biased ß2AR signaling links GRK2 upregulation to heart failure.

RATIONALE: Phosphorylation of ß(2)-adrenergic receptor (ß(2)AR) by a family of serine/threonine kinases known as G protein-coupled receptor kinase (GRK) and protein kinase A (PKA) is a critical determinant of cardiac function. Upregulation of G protein-coupled receptor kinase 2 (GRK2) is a well-established causal factor of heart failure, but the underlying mechanism is poorly understood. OBJECTIVE: We sought to determine the relative contribution of PKA- and GRK-mediated phosphorylation of ß(2)AR to the receptor coupling to G(i) signaling that attenuates cardiac reserve and contributes to the pathogenesis of heart failure in response to pressure overload. METHODS AND RESULTS: Overexpression of GRK2 led to a G(i)-dependent decrease of contractile response to ßAR stimulation in cultured mouse cardiomyocytes and in vivo. Importantly, cardiac-specific transgenic overexpression of a mutant ß(2)AR lacking PKA phosphorylation sites (PKA-TG) but not the wild-type ß(2)AR (WT-TG) or a mutant ß(2)AR lacking GRK sites (GRK-TG) led to exaggerated cardiac response to pressure overload, as manifested by markedly exacerbated cardiac maladaptive remodeling and failure and early mortality. Furthermore, inhibition of G(i) signaling with pertussis toxin restores cardiac function in heart failure associated with increased ß(2)AR to G(i) coupling induced by removing PKA phosphorylation of the receptor and in GRK2 transgenic mice, indicating that enhanced phosphorylation of ß(2)AR by GRK and resultant increase in G(i)-biased ß(2)AR signaling play an important role in the development of heart failure. CONCLUSIONS: Our data show that enhanced ß(2)AR phosphorylation by GRK, in addition to PKA, leads the receptor to G(i)-biased signaling, which, in turn, contributes to the pathogenesis of heart failure, marking G(i)-biased ß(2)AR signaling as a primary event linking upregulation of GRK to cardiac maladaptive remodeling, failure and cardiodepression.

Vessel ultrasound sonographic assessment of soluble receptor for advanced glycation end products efficacy in a rat balloon injury model.

OBJECTIVE: We aimed to assess the therapeutic efficacy of differentially modified soluble receptor for advanced glycation end products (sRAGE) in vivo using vessel ultrasound sonography and to compare the sonography data with those from postmortem histomorphologic analyses to have a practical reference for future clinical applications. METHODS: Vessel ultrasound sonography was performed in a sRAGE-treated rat carotid artery balloon injury model at different time points after the surgery, and therapeutic efficacy of different doses of sRAGE produced in Chinese hamster ovary cells and with different N-glycoform modifications were assessed. RESULTS: Vessel ultrasound sonography found that sRAGE produced in Chinese hamster ovary cells with complex N-glycoform modifications is highly effective, and is consistent with our recent findings in the same model assessed with histology. We also found that sonography is less sensitive than histology when a higher dose of sRAGE is administered. CONCLUSIONS: Sonograph results are consistent with those obtained from histology; that is, sRAGE produced in Chinese hamster ovary cells has significantly higher efficacy than insect cell-originated sRAGE cells.

A small erythropoietin derived non-hematopoietic peptide reduces cardiac inflammation, attenuates age associated declines in heart function and prolongs healthspan.

Background: Aging is associated with increased levels of reactive oxygen species and inflammation that disrupt proteostasis and mitochondrial function and leads to organism-wide frailty later in life. ARA290 (cibinetide), an 11-aa non-hematopoietic peptide sequence within the cardioprotective domain of erythropoietin, mediates tissue protection by reducing inflammation and fibrosis. Age-associated cardiac inflammation is linked to structural and functional changes in the heart, including mitochondrial dysfunction, impaired proteostasis, hypertrophic cardiac remodeling, and contractile dysfunction. Can ARA290 ameliorate these age-associated cardiac changes and the severity of frailty in advanced age? Methods: We conducted an integrated longitudinal (n = 48) and cross-sectional (n = 144) 15 months randomized controlled trial in which 18-month-old Fischer 344 x Brown Norway rats were randomly assigned to either receive chronic ARA290 treatment or saline. Serial echocardiography, tail blood pressure and body weight were evaluated repeatedly at 4-month intervals. A frailty index was calculated at the final timepoint (33 months of age). Tissues were harvested at 4-month intervals to define inflammatory markers and left ventricular tissue remodeling. Mitochondrial and myocardial cell health was assessed in isolated left ventricular myocytes. Kaplan-Meier survival curves were established. Mixed ANOVA tests and linear mixed regression analysis were employed to determine the effects of age, treatment, and age-treatment interactions. Results: Chronic ARA290 treatment mitigated age-related increases in the cardiac non-myocyte to myocyte ratio, infiltrating leukocytes and monocytes, pro-inflammatory cytokines, total NF-κB, and p-NF-κB. Additionally, ARA290 treatment enhanced cardiomyocyte autophagy flux and reduced cellular accumulation of lipofuscin. The cardiomyocyte mitochondrial permeability transition pore response to oxidant stress was desensitized following chronic ARA290 treatment. Concurrently, ARA290 significantly blunted the age-associated elevation in blood pressure and preserved the LV ejection fraction. Finally, ARA290 preserved body weight and significantly reduced other markers of organism-wide frailty at the end of life. Conclusion: Administration of ARA290 reduces cell and tissue inflammation, mitigates structural and functional changes within the cardiovascular system leading to amelioration of frailty and preserved healthspan.

Molecular and functional characterization of a novel cardiac-specific human tropomyosin isoform.

BACKGROUND: Tropomyosin (TM), an essential actin-binding protein, is central to the control of calcium-regulated striated muscle contraction. Although TPM1alpha (also called alpha-TM) is the predominant TM isoform in human hearts, the precise TM isoform composition remains unclear. METHODS AND RESULTS: In this study, we quantified for the first time the levels of striated muscle TM isoforms in human heart, including a novel isoform called TPM1kappa. By developing a TPM1kappa-specific antibody, we found that the TPM1kappa protein is expressed and incorporated into organized myofibrils in hearts and that its level is increased in human dilated cardiomyopathy and heart failure. To investigate the role of TPM1kappa in sarcomeric function, we generated transgenic mice overexpressing cardiac-specific TPM1kappa. Incorporation of increased levels of TPM1kappa protein in myofilaments leads to dilated cardiomyopathy. Physiological alterations include decreased fractional shortening, systolic and diastolic dysfunction, and decreased myofilament calcium sensitivity with no change in maximum developed tension. Additional biophysical studies demonstrate less structural stability and weaker actin-binding affinity of TPM1kappa compared with TPM1alpha. CONCLUSIONS: This functional analysis of TPM1kappa provides a possible mechanism for the consequences of the TM isoform switch observed in dilated cardiomyopathy and heart failure patients.

Beta 1-adrenergic receptor polymorphisms confer differential function and predisposition to heart failure.

Catecholamines stimulate cardiac contractility through beta(1)-adrenergic receptors (beta(1)-ARs), which in humans are polymorphic at amino acid residue 389 (Arg/Gly). We used cardiac-targeted transgenesis in a mouse model to delineate mechanisms accounting for the association of Arg389 with human heart failure phenotypes. Hearts from young Arg389 mice had enhanced receptor function and contractility compared with Gly389 hearts. Older Arg389 mice displayed a phenotypic switch, with decreased beta-agonist signaling to adenylyl cyclase and decreased cardiac contractility compared with Gly 389 hearts. Arg389 hearts had abnormal expression of fetal and hypertrophy genes and calcium-cycling proteins, decreased adenylyl cyclase and G alpha(s) expression, and fibrosis with heart failure This phenotype was recapitulated in homozygous, end-stage, failing human hearts. In addition, hemodynamic responses to beta-receptor blockade were greater in Arg389 mice, and homozygosity for Arg389 was associated with improvement in ventricular function during carvedilol treatment in heart failure patients. Thus the human Arg389 variant predisposes to heart failure by instigating hyperactive signaling programs leading to depressed receptor coupling and ventricular dysfunction, and influences the therapeutic response to beta-receptor blockade.

Striated muscle tropomyosin isoforms differentially regulate cardiac performance and myofilament calcium sensitivity.

Tropomyosin (TM) plays a central role in calcium mediated striated muscle contraction. There are three muscle TM isoforms: alpha-TM, beta-TM, and gamma-TM. alpha-TM is the predominant cardiac and skeletal muscle isoform. beta-TM is expressed in skeletal and embryonic cardiac muscle. gamma-TM is expressed in slow-twitch musculature, but is not found in the heart. Our previous work established that muscle TM isoforms confer different physiological properties to the cardiac sarcomere. To determine whether one of these isoforms is dominant in dictating its functional properties, we generated single and double transgenic mice expressing beta-TM and/or gamma-TM in the heart, in addition to the endogenously expressed alpha-TM. Results show significant TM protein expression in the betagamma-DTG hearts: alpha-TM: 36%, beta-TM: 32%, and gamma-TM: 32%. These betagamma-DTG mice do not develop pathological abnormalities; however, they exhibit a hyper contractile phenotype with decreased myofilament calcium sensitivity, similar to gamma-TM transgenic hearts. Biophysical studies indicate that gamma-TM is more rigid than either alpha-TM or beta-TM. This is the first report showing that with approximately equivalent levels of expression within the same tissue, there is a functional dominance of gamma-TM over alpha-TM or beta-TM in regulating physiological performance of the striated muscle sarcomere. In addition to the effect expression of gamma-TM has on Ca(2+) activation of the cardiac myofilaments, our data demonstrates an effect on cooperative activation of the thin filament by strongly bound rigor cross-bridges. This is significant in relation to current ideas on the control mechanism of the steep relation between Ca(2+) and tension.

An internal domain of beta-tropomyosin increases myofilament Ca(2+) sensitivity.

Tropomyosin (TM) is involved in Ca(2+)-mediated muscle contraction and relaxation in the heart. Striated muscle alpha-TM is the major isoform expressed in the heart. The expression of striated muscle beta-TM in the murine myocardium results in a decreased rate of relaxation and increased myofilament Ca(2+) sensitivity. Replacing the carboxyl terminus (amino acids 258-284) of alpha-TM with beta-TM (a troponin T-binding region) results in decreased rates of contraction and relaxation in the heart and decreased myofilament Ca(2+) sensitivity. We hypothesized that the putative internal troponin T-binding domain (amino acids 175-190) of beta-TM may be responsible for the increased myofilament Ca(2+) sensitivity observed when the entire beta-TM is expressed in the heart. To test this hypothesis, we generated transgenic mice that expressed chimeric TM containing beta-TM amino acids 175-190 in the backbone of alpha-TM (amino acids 1-174 and 191-284). These mice expressed 16-57% chimeric TM and did not develop cardiac hypertrophy or any other morphological changes. Physiological analysis showed that these hearts exhibited decreased rates of contraction and relaxation and a positive response to isoproterenol. Skinned fiber bundle analyses showed a significant increase in myofilament Ca(2+) sensitivity. Biophysical experiments demonstrated that the exchanged amino acids did not influence the flexibility of the TM. This is the first study to demonstrate that a specific domain within TM can increase the Ca(2+) sensitivity of the thin filament and affect sarcomeric performance. Furthermore, these results enhance the understanding of why TM mutations associated with familial hypertrophic cardiomyopathy demonstrate increased myofilament sensitivity to Ca(2+).

Targeted disruption of the voltage-dependent calcium channel alpha2/delta-1-subunit.

Cardiac L-type voltage-dependent Ca(2+) channels are heteromultimeric polypeptide complexes of alpha(1)-, alpha(2)/delta-, and beta-subunits. The alpha(2)/delta-1-subunit possesses a stereoselective, high-affinity binding site for gabapentin, widely used to treat epilepsy and postherpetic neuralgic pain as well as sleep disorders. Mutations in alpha(2)/delta-subunits of voltage-dependent Ca(2+) channels have been associated with different diseases, including epilepsy. Multiple heterologous coexpression systems have been used to study the effects of the deletion of the alpha(2)/delta-1-subunit, but attempts at a conventional knockout animal model have been ineffective. We report the development of a viable conventional knockout mouse using a construct targeting exon 2 of alpha(2)/delta-1. While the deletion of the subunit is not lethal, these animals lack high-affinity gabapentin binding sites and demonstrate a significantly decreased basal myocardial contractility and relaxation and a decreased L-type Ca(2+) current peak current amplitude. This is a novel model for studying the function of the alpha(2)/delta-1-subunit and will be of importance in the development of new pharmacological therapies.

Dilated cardiomyopathy mutant tropomyosin mice develop cardiac dysfunction with significantly decreased fractional shortening and myofilament calcium sensitivity.

Mutations in striated muscle alpha-tropomyosin (alpha-TM), an essential thin filament protein, cause both dilated cardiomyopathy (DCM) and familial hypertrophic cardiomyopathy. Two distinct point mutations within alpha-tropomyosin are associated with the development of DCM in humans: Glu40Lys and Glu54Lys. To investigate the functional consequences of alpha-TM mutations associated with DCM, we generated transgenic mice that express mutant alpha-TM (Glu54Lys) in the adult heart. Results showed that an increase in transgenic protein expression led to a reciprocal decrease in endogenous alpha-TM levels, with total myofilament TM protein levels remaining unaltered. Histological and morphological analyses revealed development of DCM with progression to heart failure and frequently death by 6 months. Echocardiographic analyses confirmed the dilated phenotype of the heart with a significant decrease in the left ventricular fractional shortening. Work-performing heart analyses showed significantly impaired systolic, and diastolic functions and the force measurements of cardiac myofibers revealed that the myofilaments had significantly decreased Ca(2+) sensitivity and tension generation. Real-time RT-PCR quantification demonstrated an increased expression of beta-myosin heavy chain, brain natriuretic peptide, and skeletal actin and a decreased expression of the Ca(2+) handling proteins sarcoplasmic reticulum Ca(2+)-ATPase and ryanodine receptor. Furthermore, our study also indicates that the alpha-TM54 mutation decreases tropomyosin flexibility, which may influence actin binding and myofilament Ca(2+) sensitivity. The pathological and physiological phenotypes exhibited by these mice are consistent with those seen in human DCM and heart failure. As such, this is the first mouse model in which a mutation in a sarcomeric thin filament protein, specifically TM, leads to DCM.

Monoclonal Antibody to Marinobufagenin Downregulates TGFß Profibrotic Signaling in Left Ventricle and Kidney and Reduces Tissue Remodeling in Salt-Sensitive Hypertension.

Background Elevated levels of an endogenous Na/K-ATPase inhibitor marinobufagenin accompany salt-sensitive hypertension and are implicated in cardiac fibrosis. Immunoneutralization of marinobufagenin reduces blood pressure in Dahl salt-sensitive (Dahl-S) rats. The effect of the anti-marinobufagenin monoclonal antibody on blood pressure, left ventricular (LV) and renal remodeling, and gene expression were investigated in hypertensive Dahl-S rats. Methods and Results Dahl-S rats were fed high NaCl (8%, HS; n=14) or low NaCl (0.1%, LS; n=14) diets for 8 weeks. Animals were administered control antibody (LS control antibody, LSC; HS control antibody, HSC; n=7 per group) or anti-marinobufagenin antibody once on week 7 of diet intervention (n=7 per group). Levels of marinobufagenin, LV, and kidney mRNAs and proteins implicated in profibrotic signaling were assessed. Systolic blood pressure was elevated (211±8 versus 133±3 mm Hg, P<0.01), marinobufagenin increased 2-fold in plasma (P<0.05) and 5-fold in urine (P<0.01), LV and kidney weights increased, and levels of LV collagen-1 rose 3.5-fold in HSC versus LSC. Anti-marinobufagenin antibody treatment decreased systolic blood pressure by 24 mm Hg (P<0.01) and reduced organ weights and level of LV collagen-1 (P<0.01) in hypertensive Dahl salt-sensitive rats with anti-marinobufagenin antibody versus HSC. The expression of genes related to transforming growth factor-ß-dependent signaling was upregulated in the left ventricles and kidneys in HSC versus LSC groups and became downregulated following administration of anti-marinobufagenin antibody to hypertensive Dahl-S rats. Marinobufagenin also activated transforming growth factor-ß signaling in cultured ventricular myocytes from Dahl-S rats. Conclusions Immunoneutralization of heightened marinobufagenin levels in hypertensive Dahl-S rats resulted in a downregulation of genes implicated in transforming growth factor-ß pathway, which indicates that marinobufagenin is an activator of profibrotic transforming growth factor-ß-dependent signaling in salt-sensitive hypertension.

G protein, phosphorylated-GATA4 and VEGF expression in the hearts of transgenic mice overexpressing ß1- and ß2-adrenergic receptors.

ß1- and ß2-adrenergic receptors (ARs) regulate cardiac contractility, calcium handling and protein phosphorylation. The present study aimed to examine the expression levels of vascular endothelial growth factor A (VEGF­A) and several G proteins, and the phosphorylation of transcription factor GATA binding protein 4 (GATA4), by western blot analysis, using isolated hearts from 6 month­old transgenic (TG) mice that overexpress ß1AR or ß2AR. Cardiac contractility/relaxation and heart rate was increased in both ß1AR TG and ß2AR TG mouse hearts compared with wild type; however, no significant differences were observed between the ß1­ and ß2AR TG mouse hearts. Protein expression levels of inhibitory guanine nucleotide­binding protein (Gi) 2, Gi3 and G­protein­coupled receptor kinase 2 were upregulated in both TG mice, although the upregulation of Gi2 was more prominent in the ß2AR TG mice. VEGF­A expression levels were also increased in both TG mice, and were highest in the ß1AR TG mice. In addition, the levels of phosphorylated­GATA4 expression were increased in ß1­ and ß2AR TG mice. In conclusion, the present study demonstrated that cardiac contractility/relaxation and heart rate is increased in ß1AR TG and ß2AR TG mice, and indicated that this increase may be related to the overexpression of G proteins and G­protein­associated proteins.

Effects of alpha1-adrenergic stimulation on normal and hypertrophied mouse hearts. Relation to caveolin-3 expression.

BACKGROUND: Modulation of the transduction efficiency through G-protein coupled receptors, caused by external stimulation, is essential in designing antihypertrophic treatment strategies in the dysfunctional heart. We compared protein-kinase C (PKC)-dependent regulation of positive inotropic effect via alpha1-adrenoreceptor (ADR)/Gq protein in hyperdynamic versus hypertrophied myocardium. METHODS: Inotropic (work performing isolated heart) and cellular effects of alpha1-adrenoreceptor stimulation were studied in nontransgenic (Ntg) and transgenic (Tg) mice with cardiac specific overexpression of L-type voltage-dependent calcium channels (L-type VDCC). RESULTS: Transgenic hyperdynamic and hypertrophic myocardium (due to overexpression of the L-type VDCC alpha1 subunit) were characterized by a lack of positive inotropic effect (PIE) to alpha1-ADR stimulation with phenylephrine (PE), as compared to a positive response in Ntg hearts. This was partially restored by PKC inhibition with chelerythrine and staurosporine only at the hyperdynamic stage. The inability of PKC inhibition to increase positive inotropy was associated with markedly decreased cardiac-specific caveolin-3 expression, and no changes in Galphaq, PLC-beta1, caveolin-1 and alpha1-adrenoreceptor expression. CONCLUSION: In the hyperdynamic myocardium, PKC activation may be one of the switches responsible for an impaired alpha1-adrenergic positive inotropic response. In the hypertrophied myocardium, the interruption of the transduction from Galphaq-protein coupled receptors to downstream effectors may be due to the down-regulation of caveolin-3 expression.

Cardiac function and electrical remodeling of the calcineurin-overexpressed transgenic mouse.

OBJECTIVE: To study the specificity of contractile phenotype and electrophysiological remodeling in transgenic (Tg) mice with cardiac directed calcineurin (phosphatase 2B) overexpression and evaluate a possible negative role of chronically activated calcineurin in beta-adrenergic mediated contractile response. METHODS: The patch-clamp technique was used to characterize electrophysiological properties of action potentials and inward rectifier (I(K1)), and transient outward potassium currents (I(to)). The analysis of the contractile performance was carried out on isolated retrograde perfused hearts at constant aortic pressure. RESULTS: Tg mice demonstrated a hypercontractile phenotype characterized by a profound beta-adrenergic hypo-responsiveness at 2.0 mM [Ca2+](o). Transgenic cardiomyocytes showed marked action potential prolongation (209% in APD(90)) with increased I(to,peak) and I(sus) and decreased protein expression level of Kv1.5 and Kv2.1. Lowering [Ca2+](o) to 0.75 mM restored the beta-adrenergic response, indicating that the calcineurin/calmodulin/adenylyl cyclase (AC) pathway may not be directly responsible for the blunted beta-adrenoreceptor mediated inotropism. CONCLUSIONS: Calcineurin overexpression leads to development of a hyperdynamic phenotype with a cellular profile of increased calcium influx. This type of functional hypertrophic remodeling is accompanied by a negative feedback regulation between increased calcium handling and beta-adrenergic contractile activation.

The N-glycoform of sRAGE is the key determinant for its therapeutic efficacy to attenuate injury-elicited arterial inflammation and neointimal growth.

UNLABELLED: Signaling of the receptor for advanced glycation end products (RAGE) has been implicated in the development of injury-elicited vascular complications. Soluble RAGE (sRAGE) acts as a decoy of RAGE and has been used to treat pathological vascular conditions in animal models. However, previous studies used a high dose of sRAGE produced in insect Sf9 cells (sRAGE(Sf9))and multiple injections to achieve the therapeutic outcome. Here, we explore whether modulation of sRAGE N-glycoform impacts its bioactivity and augments its therapeutic efficacy. We first profiled carbohydrate components of sRAGE produced in Chinese hamster Ovary cells (sRAGE(CHO)) to show that a majority of its N-glycans belong to sialylated complex types that are not shared by sRAGE(Sf9). In cell-based NF-κB activation and vascular smooth muscle cell (VSMC) migration assays, sRAGE(CHO) exhibited a significantly higher bioactivity relative to sRAGE(Sf9) to inhibit RAGE alarmin ligand-induced NF-κB activation and VSMC migration. We next studied whether this N-glycoform-associated bioactivity of sRAGE(CHO) is translated to higher in vivo therapeutic efficacy in a rat carotid artery balloon injury model. Consistent with the observed higher bioactivity in cell assays, sRAGE(CHO) significantly reduced injury-induced neointimal growth and the expression of inflammatory markers in injured vasculature. Specifically, a single dose of 3 ng/g of sRAGE(CHO) reduced neointimal hyperplasia by over 70%, whereas the same dose of sRAGE(Sf9) showed no effect. The administered sRAGE(CHO) is rapidly and specifically recruited to the injured arterial locus, suggesting that early intervention of arterial injury with sRAGE(CHO) may offset an inflammatory circuit and reduce the ensuing tissue remodeling. Our findings showed that the N-glycoform of sRAGE is the key determinant underlying its bioactivity and thus is an important glycobioengineering target to develop a highly potent therapeutic sRAGE for future clinical applications. KEY MESSAGE: The specific N-glycoform modification is the key underlying sRAGE bioactivity Markedly reduced sRAGE dose to attenuate neointimal hyperplasia and inflammation Provide a molecular target for glycobioengineering of sRAGE as a therapeutic protein Blocking RAGE alarmin ligands during acute injury phase offsets neointimal growth.