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.

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.

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.

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.

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.

Presynaptic stimulus-release and postsynaptic compensatory changes in mice lacking the N-type calcium channel α(1B)-subunit.

N-type (Ca(v)2.2) voltage-dependent calcium channels (VDCC) play an important role in presynaptic neurotransmitter release in the autonomic nervous system and may be clinically relevant in the treatment of cardiovascular diseases. The physiological impact of N-type VDCC ablation on cardiac function, stimulus-release coupling and cardiac autonomic regulation was studied using mice deficient in the α(1B) subunit of the N-type channel (N-type-/-).The positive inotropic effect (increase in +dP/dt) secondary to high frequency field stimulation (HFFS), mediated by the sympathetic nervous system, was decreased by 33 ± 12.6% in N-type-/- versus 89 ± 11.4% in Wild-Type (WT)(P<0.01), whereas the negative inotropic response (decrease in +dP/dt) following HFFS in the presence of propranolol, mediated by the parasympathetic nervous system, was similar to that in Wild-type (WT) animals 34 ± 5.0% and 35 ± 5.4%, respectively. There were no changes in the postsynaptic ß-adrenergic responsiveness, ß-adrenoreceptor density or adenylyl cyclase activity. N-type-/- hearts demonstrated an increased contractile response to α(1)-adrenoreceptor (α(1)-ADR) stimulation with 10(-5)M phenylephrine in the presence of the ß-blocker propranolol, which might be attributed to an increased expression of PLCß1. Protein abundance of other signal transducers for α(1) ADR transduction protein was not changed in the N-type-/- hearts. These results suggest that selective impairment of sympathetic inflow does not modulate postsynaptic ß-adrenergic responsiveness, but causes increased functional response to α(1)-adrenergic stimulation.

Inhibition of protein kinase C alpha improves myocardial beta-adrenergic receptor signaling and ventricular function in a model of myocardial preservation.

OBJECTIVE: The specific effect of protein kinase C alpha, the primary ventricular calcium-dependent protein kinase C isoform, on myocardial protection is unclear. The objective of this study was to determine the role of protein kinase C alpha in myocardial protection and recovery of function after cardioplegic arrest, cold preservation, and normothermic reperfusion, as relevant to cardiac transplantation. METHODS: We used an ex vivo murine model, and hearts were arrested with cold crystalloid cardioplegia or saline as a control and maintained at 4 degrees C for 4 hours. This was followed by normothermic reperfusion for 90 minutes. Transgenic hearts with cardiac-specific activation or inhibition of protein kinase C alpha were then studied to specifically examine the effects of protein kinase C alpha on myocardial preservation in this model. RESULTS: Cardioplegic arrest with University of Wisconsin solution led to significantly improved postreperfusion hemodynamics and inhibition of myocardial protein kinase C alpha activity relative to that seen in saline-treated control hearts. Beta-adrenergic receptor signaling was also preserved with University of Wisconsin solution. Transgenic hearts with enhanced protein kinase C alpha activity had poor postreperfusion hemodynamics, impaired beta-adrenergic receptor signaling, and increased G protein-coupled receptor kinase 2 activity compared with those seen in nontransgenic control hearts. In contrast, transgenic hearts with inhibited protein kinase C alpha activity had even better myocardial protection relative to control hearts and preserved beta-adrenergic receptor signaling. CONCLUSIONS: Current techniques of myocardial preservation are associated with inhibition of protein kinase C alpha activity and maintenance of intact beta-adrenergic receptor signaling. Activation of protein kinase C alpha leads to enhanced beta-adrenergic receptor desensitization and impaired signaling and ventricular function as a result of increased G protein-coupled receptor kinase 2 activity. This is a novel in vivo mechanism of G protein-coupled receptor kinase 2 activation. Strategies to specifically inhibit these kinases might improve long-term myocardial protection.

Ablation of sarcolipin enhances sarcoplasmic reticulum calcium transport and atrial contractility.

Sarcolipin is a novel regulator of cardiac sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and is expressed abundantly in atria. In this study we investigated the physiological significance of sarcolipin in the heart by generating a mouse model deficient for sarcolipin. The sarcolipin-null mice do not show any developmental abnormalities or any cardiac pathology. The absence of sarcolipin does not modify the expression level of other Ca2+ handling proteins, in particular phospholamban, and its phosphorylation status. Calcium uptake studies revealed that, in the atria, ablation of sarcolipin resulted in an increase in the affinity of the SERCA pump for Ca2+ and the maximum velocity of Ca2+ uptake rates. An important finding is that ablation of sarcolipin resulted in an increase in atrial Ca2+ transient amplitudes, and this resulted in enhanced atrial contractility. Furthermore, atria from sarcolipin-null mice showed a blunted response to isoproterenol stimulation, implicating sarcolipin as a mediator of beta-adrenergic responses in atria. Our study documented that sarcolipin is a key regulator of SERCA2a in atria. Importantly, our data demonstrate the existence of distinct modulators for the SERCA pump in the atria and ventricles.

Myocardial beta1-adrenergic receptor polymorphisms affect functional recovery after ischemic injury.

Association studies suggest beta(1)-adrenergic receptor (beta(1)-AR) polymorphisms are disease modifiers in heart failure. The Arg389 variant has increased coupling to G(s) in transfected cells and evokes enhanced ventricular function in transgenic mice. Here, we assessed the differential effects of the human Gly389 and Arg389 beta(1)-AR polymorphisms on myocardial recovery after ischemic injury. Function was studied in transgenic mice with cardiac-specific expression of either human Gly389 or Arg389 beta(1)-AR at baseline and after 20 min of ex vivo ischemia and reperfusion (I/R). In 3-mo-old mice of either genotype, there was poor recovery after I/R (approximately 38% vs. approximately 68% for nontransgenic). Paradoxically, at 6 mo of age, functional recovery remained severely depressed in Gly389 hearts (approximately 32%) but was similar to nontransgenic for Arg389 hearts (approximately 60%). In Arg389 hearts, agonist-promoted adenylyl cyclase activities were depressed by approximately 35% at 6 mo of age, and G protein-coupled receptor kinase (GRK) activity was increased by approximately twofold compared with Gly389. Furthermore, I/R evoked an approximately threefold increase in ERK2 phosphorylation in Arg389 but an approximately twofold decrease in Gly389 hearts. Individually, these changes have been shown to mitigate I/R injury; thus the Arg389-beta(1)-AR uniquely evokes specialized pathways that act to protect against I/R injury. The improved recovery of function after I/R in Arg389 hearts relative to Gly389 appears to be due to an adaptive multimechanism program with allele-specific alterations in receptor signaling, GRK activity, and ERK2. Thus genetic variation of the human beta(1)-AR may play a role in cardiac functional recovery after ischemic injury.

Targeted overexpression of sarcolipin in the mouse heart decreases sarcoplasmic reticulum calcium transport and cardiac contractility.

The role of sarcolipin (SLN) in cardiac physiology was critically evaluated by generating a transgenic (TG) mouse model in which the SLN to sarco(endoplasmic)reticulum (SR) Ca(2+) ATPase (SERCA) ratio was increased in the ventricle. Overexpression of SLN decreases SR calcium transport function and results in decreased calcium transient amplitude and rate of relaxation. SLN TG hearts exhibit a significant decrease in rates of contraction and relaxation when assessed by ex vivo work-performing heart preparations. Similar results were also observed with muscle preparations and myocytes from SLN TG ventricles. Interestingly, the inhibitory effect of SLN was partially relieved upon high dose of isoproterenol treatment and stimulation at high frequency. Biochemical analyses show that an increase in SLN level does not affect PLB levels, monomer to pentamer ratio, or its phosphorylation status. No compensatory changes were seen in the expression of other calcium-handling proteins. These studies suggest that the SLN effect on SERCA pump is direct and is not mediated through increased monomerization of PLB or by a change in PLB phosphorylation status. We conclude that SLN is a novel regulator of SERCA pump activity, and its inhibitory effect can be reversed by beta-adrenergic agonists.

Calcium cycling, historic overview and perspectives. Role for autonomic nervous system regulation.

The human heart proceeds from a relaxed state (diastole) to a fully contracted state (systole) and recovery in 600ms. During this period, Ca(2+) inside the myocardial cell rises from about 10nM to about 100nM and returns to the former. The contractile-relaxation cycle is tightly coupled to the Ca(2+)transient. In the normal physiological state, the autonomic nervous system (ANS) plays a major role in the regulation of cardiac function and important changes occur in diseases of the heart. Sympathetic overdrive is a major determinant of the critical transition from initial compensatory hypertrophy to decompensated failure. Cardiac myocytes from failing hearts are characterized by a number of abnormalities in excitation-contraction coupling, that are a direct consequence of beta-adrenergic signaling defects. Although desensitized in cardiac hypertrophy and failure, the beta-adrenergic signaling pathway retains receptor capacity, a characteristic that is used in therapeutic approaches. There are several putative Ca(2+)-dependent pathways that exert counterbalancing negative regulation over cAMP-dependent positive inotropic effect and may represent potential targets for contractile stimulation. This review is focused on the interactions between sympathetic drive and aspects of calcium signaling in the heart.