Periodic injections of Relaxin 2, its pharmacokinetics and remodeling of rat hearts.

Relaxin-2 (RLX), a critical hormone in pregnancy, has been investigated as a therapy for heart failure. In most studies, the peptide was delivered continuously, subcutaneously for 2 weeks in animals or intravenously for 2-days in human subjects, for stable circulating [RLX]. However, pulsatile hormone levels may better uncover the normal physiology. This premise was tested by subcutaneously injecting Sprague Dawley rats (250 g, N = 2 males, 2 females/group) with human RLX (0, 30, 100, or 500 µg/kg), every 12 h for 1 day, then measuring changes in Nav1.5, connexin43, and ß-catenin, 24 h later. Pulsatile RLX was measured by taking serial blood draws, post-injection. After an injection, RLX reached a peak in âˆ¼ 60 min, fell to 50 % in 5-6 h; injections of 0, 30, 100 or 500 µg/kg yielded peak levels of 0, 11.26 ± 3.52, 58.33 ± 16.10, and 209.42 ± 29.04 ng/ml and residual levels after 24-hrs of 0, 4.9, 45.1 and 156 pg/ml, respectively. The 30 µg/kg injections had no effect and 100 µg/kg injections increased Nav1.5 (25 %), Cx43 (30 %) and ß-catenin (90 %). The 500 µg/kg injections also increased Nav1.5 and Cx43 but were less effective at upregulating ß-catenin (up by 25 % vs. 90 %). Periodic injections of 100 µg/kg were highly effective at increasing the expression of Nav1.5 and Cx43 which are key determinants of conduction velocity in the heart and the suppression of arrhythmias. Periodic RLX is effective at eliciting changes in cardiac protein expression and may be a better strategy for its longer-term delivery in the clinical setting.

Guide to assembling a successful K99/R00 application.

The National Institutes of Health's (NIH) K99/R00 Pathway to Independence Award offers promising postdoctoral researchers and clinician-scientists an opportunity to receive research support at both the mentored and the independent levels with the goal of facilitating a timely transition to a tenure-track faculty position. This transitional program has been generally successful, with most K99/R00 awardees successfully securing R01-equivalent funding by the end of the R00 period. However, often highly promising proposals fail because of poor grantsmanship. This overview provides guidance from the perspective of long-standing members of the National Heart, Lung, and Blood Institute's Mentored Transition to Independence study section for the purpose of helping mentors and trainees regarding how best to assemble competitive K99/R00 applications.

Cardiomyocyte-Targeting Peptide to Deliver Amiodarone.

BACKGROUND: Amiodarone is underutilized due to significant off-target toxicities. We hypothesized that targeted delivery to the heart would lead to the lowering of the dose by utilizing a cardiomyocyte-targeting peptide (CTP), a cell-penetrating peptide identified by our prior phage display work. METHODS: CTP was synthesized thiolated at the N-terminus, conjugated to amiodarone via Schiff base chemistry, HPLC purified, and confirmed with MALDI/TOF. The stability of the conjugate was assessed using serial HPLCs. Guinea pigs (GP) were injected intraperitoneally daily with vehicle (7 days), amiodarone (7 days; 80 mg/kg), CTP-amiodarone (5 days; 26.3 mg/kg), or CTP (5 days; 17.8 mg/kg), after which the GPs were euthanized, and the hearts were excised and perfused on a Langendorff apparatus with Tyrode's solution and blebbistatin (5 µM) to minimize the contractions. Voltage (RH237) and Ca2+-indicator dye (Rhod-2/AM) were injected, and fluorescence from the epicardium split and was captured by two cameras at 570-595 nm for the cytosolic Ca2+ and 610-750 nm wavelengths for the voltage. Subsequently, the hearts were paced at 250 ms with programmed stimulation to measure the changes in the conduction velocities (CV), action potential duration (APD), and Ca2+ transient durations at 90% recovery (CaTD90). mRNA was extracted from all hearts, and RNA sequencing was performed with results compared to the control hearts. RESULTS: The CTP-amiodarone remained stable for up to 21 days at 37 °C. At ~1/15th of the dose of amiodarone, the CTP-amiodarone decreased the CV in hearts significantly compared to the control GPs (0.92 ± 0.05 vs. 1.00 ± 0.03 ms, p = 0.0007), equivalent to amiodarone alone (0.87 ± 0.08 ms, p = 0.0003). Amiodarone increased the APD (192 ± 5 ms vs. 175 ± 8 ms for vehicle, p = 0.0025), while CTP-amiodarone decreased it significantly (157 ± 16 ms, p = 0.0136), similar to CTP alone (155 ± 13 ms, p = 0.0039). Both amiodarone and CTP-amiodarone significantly decreased the calcium transients compared to the controls. CTP-amiodarone and CTP decreased the CaTD90 to an extent greater than amiodarone alone (p < 0.001). RNA-seq showed that CTP alone increased the expression of DHPR and SERCA2a, while it decreased the expression of the proinflammatory genes, NF-kappa B, TNF-α, IL-1ß, and IL-6. CONCLUSIONS: Our data suggest that CTP can deliver amiodarone to cardiomyocytes at ~1/15th the total molar dose of the amiodarone needed to produce a comparable slowing of CVs. The ability of CTP to decrease the AP durations and CaTD90 may be related to its increase in the expression of Ca-handling genes, which merits further study.

Conjugation of amiodarone to a novel cardiomyocyte cell penetrating peptide for potential targeted delivery to the heart.

Modern medicine has developed a myriad of therapeutic drugs against a wide range of human diseases leading to increased life expectancy and better quality of life for millions of people. Despite the undeniable benefit of medical advancements in pharmaceutical technology, many of the most effective drugs currently in use have serious limitations such as off target side effects resulting in systemic toxicity. New generations of specialized drug constructs will enhance targeted therapeutic efficacy of existing and new drugs leading to safer and more effective treatment options for a variety of human ailments. As one of the most efficient drugs known for the treatment of cardiac arrhythmia, Amiodarone presents the same conundrum of serious systemic side effects associated with long term treatment. In this article we present the synthesis of a next-generation prodrug construct of amiodarone for the purpose of advanced targeting of cardiac arrhythmias by delivering the drug to cardiomyocytes using a novel cardiac targeting peptide, a cardiomyocyte-specific cell penetrating peptide. Our in vivo studies in guinea pigs indicate that cardiac targeting peptide-amiodarone conjugate is able to have similar effects on calcium handling as amiodarone at 1/15th the total molar dose of amiodarone. Further studies are warranted in animal models of atrial fibrillation to show efficacy of this conjugate.

Conduction delays across the specialized conduction system of the heart: Revisiting atrioventricular node (AVN) and Purkinje-ventricular junction (PVJ) delays.

Background and significance: The specialized conduction system (SCS) of the heart was extensively studied to understand the synchronization of atrial and ventricular contractions, the large atrial to His bundle (A-H) delay through the atrioventricular node (AVN), and delays between Purkinje (P) and ventricular (V) depolarization at distinct junctions (J), PVJs. Here, we use optical mapping of perfused rabbit hearts to revisit the mechanism that explains A-H delay and the role of a passive electrotonic step-delay at the boundary between atria and the AVN. We further visualize how the P anatomy controls papillary activation and valve closure before ventricular activation. Methods: Rabbit hearts were perfused with a bolus (100-200 µl) of a voltage-sensitive dye (di4ANEPPS), blebbistatin (10-20 µM for 20 min) then the right atrial appendage and ventricular free-wall were cut to expose the AVN, P fibers (PFs), the septum, papillary muscles, and the endocardium. Fluorescence images were focused on a CMOS camera (SciMedia) captured at 1K-5 K frames/s from 100 × 100 pixels. Results: AP propagation across the AVN-His (A-H) exhibits distinct patterns of delay and conduction blocks during S1-S2 stimulation. Refractory periods were 81 ± 9, 90 ± 21, 185 ± 15 ms for Atrial, AVN, and His, respectively. A large delay (>40 ms) occurs between atrial and AVN activation that increased during rapid atrial pacing contributing to the development of Wenckebach periodicity followed by delays within the AVN through slow or blocked conduction. The temporal resolution of the camera allowed us to identify PVJs by detecting doublets of AP upstrokes. PVJ delays were heterogeneous, fastest in PVJ that immediately trigger ventricular APs (3.4 ± 0.8 ms) and slow in regions where PF appear insulated from the neighboring ventricular myocytes (7.8 ± 2.4 ms). Insulated PF along papillary muscles conducted APs (>2 m/s), then triggered papillary muscle APs (<1 m/s), followed by APs firing of septum and endocardium. The anatomy of PFs and PVJs produced activation patterns that control the sequence of contractions ensuring that papillary contractions close the tricuspid valve 2-5 ms before right ventricular contractions. Conclusions: The specialized conduction system can be accessed optically to investigate the electrical properties of the AVN, PVJ and activation patterns in physiological and pathological conditions.

Cardiomyocyte Targeting Peptide to Deliver Amiodarone.

Background: Amiodarone is underutilized due to significant off-target toxicities. We hypothesized that targeted delivery to the heart would lead to lowering of dose by utilizing a cardiomyocyte targeting peptide (CTP), a cell penetrating peptide identified by our prior phage display work. Methods: CTP was synthesized thiolated at the N-terminus, conjugated to amiodarone via Schiff base chemistry, HPLC purified and confirmed with MALDI/TOF. Stability of the conjugate was assessed using serial HPLCs. Guinea pigs (GP) were injected intraperitoneally daily with vehicle (7 days), amiodarone (7 days; 80mg/Kg), CTP-amiodarone (5 days;26.3mg/Kg), or CTP (5 days; 17.8mg/Kg), after which GPs were euthanized, hearts excised, perfused on a Langendorff apparatus with Tyrode's solution and blebbistatin (5µM) to minimize contractions. Voltage (RH237) and Ca 2+ -indicator dye (Rhod-2/AM) were injected, fluorescence from the epicardium split and focused on two cameras capturing at 570-595nm for cytosolic Ca 2+ and 610-750nm wavelengths for voltage. Subsequently, hearts were paced at 250ms with programmed stimulation to measure changes in conduction velocities (CV), action potential duration (APD) and Ca 2+ transient durations at 90% recovery (CaTD 90 ). mRNA was extracted from all hearts and RNA sequencing performed with results compared to control hearts. Results: CTP-amiodarone remained stable for up to 21 days at 37°C. At ∼1/15 th of the dose of amiodarone, CTP-amiodarone decreased CV in hearts significantly compared to control GPs (0.92±0.05 vs. 1.00±0.03m/s, p=0.0007), equivalent to amiodarone alone (0.87±0.08ms, p=0.0003). Amiodarone increased APD (192±5ms vs. 175±8ms for vehicle, p=0.0025), while CTP-amiodarone decreased it significantly (157±16ms, p=0.0136) similar to CTP alone (155±13ms, p=0.0039). Both amiodarone and CTP-amiodarone significantly decreased calcium transients compared to controls. CTP-amiodarone and CTP decreased CaTD 90 to an extent greater than amiodarone alone (p<0.001). RNA-seq showed that CTP alone increased the expression of DHPR and SERCA2a, while decreasing expression of proinflammatory genes NF-kappa B, TNF-α, IL-1ß, and IL-6. Conclusions: Our data suggests that CTP can deliver amiodarone to cardiomyocytes at ∼1/15 th the total molar dose of amiodarone needed to produce comparable slowing of CVs. The ability of CTP to decrease AP durations and CaTD 90 may be related to its increase in expression of Ca-handling genes, and merits further study.

Loss of cardiomyocyte CYB5R3 impairs redox equilibrium and causes sudden cardiac death.

Sudden cardiac death (SCD) in patients with heart failure (HF) is allied with an imbalance in reduction and oxidation (redox) signaling in cardiomyocytes; however, the basic pathways and mechanisms governing redox homeostasis in cardiomyocytes are not fully understood. Here, we show that cytochrome b5 reductase 3 (CYB5R3), an enzyme known to regulate redox signaling in erythrocytes and vascular cells, is essential for cardiomyocyte function. Using a conditional cardiomyocyte-specific CYB5R3-knockout mouse, we discovered that deletion of CYB5R3 in male, but not female, adult cardiomyocytes causes cardiac hypertrophy, bradycardia, and SCD. The increase in SCD in CYB5R3-KO mice is associated with calcium mishandling, ventricular fibrillation, and cardiomyocyte hypertrophy. Molecular studies reveal that CYB5R3-KO hearts display decreased adenosine triphosphate (ATP), increased oxidative stress, suppressed coenzyme Q levels, and hemoprotein dysregulation. Finally, from a translational perspective, we reveal that the high-frequency missense genetic variant rs1800457, which translates into a CYB5R3 T117S partial loss-of-function protein, associates with decreased event-free survival (~20%) in Black persons with HF with reduced ejection fraction (HFrEF). Together, these studies reveal a crucial role for CYB5R3 in cardiomyocyte redox biology and identify a genetic biomarker for persons of African ancestry that may potentially increase the risk of death from HFrEF.

High-resolution structure-function mapping of intact hearts reveals altered sympathetic control of infarct border zones.

Remodeling of injured sympathetic nerves on the heart after myocardial infarction (MI) contributes to adverse outcomes such as sudden arrhythmic death, yet the underlying structural mechanisms are poorly understood. We sought to examine microstructural changes on the heart after MI and to directly link these changes with electrical dysfunction. We developed a high-resolution pipeline for anatomically precise alignment of electrical maps with structural myofiber and nerve-fiber maps created by customized computer vision algorithms. Using this integrative approach in a mouse model, we identified distinct structure-function correlates to objectively delineate the infarct border zone, a known source of arrhythmias after MI. During tyramine-induced sympathetic nerve activation, we demonstrated regional patterns of altered electrical conduction aligned directly with altered neuroeffector junction distribution, pointing to potential neural substrates for cardiac arrhythmia. This study establishes a synergistic framework for examining structure-function relationships after MI with microscopic precision that has potential to advance understanding of arrhythmogenic mechanisms.

Cardiac natriuretic peptide deficiency sensitizes the heart to stress-induced ventricular arrhythmias via impaired CREB signalling.

AIMS: The cardiac natriuretic peptides [atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP)] are important regulators of cardiovascular physiology, with reduced natriuretic peptide (NP) activity linked to multiple human cardiovascular diseases. We hypothesized that deficiency of either ANP or BNP would lead to similar changes in left ventricular structure and function given their shared receptor affinities. METHODS AND RESULTS: We directly compared murine models deficient of ANP or BNP in the same genetic backgrounds (C57BL6/J) and environments. We evaluated control, ANP-deficient (Nppa-/-) or BNP-deficient (Nppb-/-) mice under unstressed conditions and multiple forms of pathological myocardial stress. Survival, myocardial structure, function and electrophysiology, tissue histology, and biochemical analyses were evaluated in the groups. In vitro validation of our findings was performed using human-derived induced pluripotent stem cell cardiomyocytes (iPS-CMs). In the unstressed state, both ANP- and BNP-deficient mice displayed mild ventricular hypertrophy which did not increase up to 1 year of life. NP-deficient mice exposed to acute myocardial stress secondary to thoracic aortic constriction (TAC) had similar pathological myocardial remodelling but a significant increase in sudden death. We discovered that the NP-deficient mice are more susceptible to stress-induced ventricular arrhythmias using both in vivo and ex vivo models. Mechanistically, deficiency of either ANP or BNP led to reduced myocardial cGMP levels and reduced phosphorylation of the cAMP response element-binding protein (CREBS133) transcriptional regulator. Selective CREB inhibition sensitized wild-type hearts to stress-induced ventricular arrhythmias. ANP and BNP regulate cardiomyocyte CREBS133 phosphorylation through a cGMP-dependent protein kinase 1 (PKG1) and p38 mitogen-activated protein kinase (p38 MAPK) signalling cascade. CONCLUSIONS: Our data show that ANP and BNP act in a non-redundant fashion to maintain myocardial cGMP levels to regulate cardiomyocyte p38 MAPK and CREB activity. Cardiac natriuretic peptide deficiency leads to a reduction in CREB signalling which sensitizes the heart to stress-induced ventricular arrhythmias.

Relaxin Inhibits Ventricular Arrhythmia and Asystole in Rats With Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) leads to right ventricular cardiomyopathy and cardiac dysfunctions where in the clinical setting, cardiac arrest is the likely cause of death, in ~70% of PAH patients. We investigated the cardiac phenotype of PAH hearts and tested the hypothesis that the insulin-like hormone, Relaxin could prevent maladaptive cardiac remodeling and protect against cardiac dysfunctions in a PAH animal model. PAH was induced in rats with sugen (20 mg/kg), hypoxia then normoxia (3-weeks/each); relaxin (RLX = 0, 30 or 400 µg/kg/day, n ≥ 6/group) was delivered subcutaneously (6-weeks) with implanted osmotic mini-pumps. Right ventricle (RV) hemodynamics and Doppler-flow measurements were followed by cardiac isolation, optical mapping, and arrhythmia phenotype. Sugen-hypoxia (SuHx) treated rats developed PAH characterized by higher RV systolic pressures (50 ± 19 vs. 22 ± 5 mmHg), hypertrophy, reduced stroke volume, ventricular fibrillation (VF) (n = 6/11) and bradycardia/arrest (n = 5/11); both cardiac phenotypes were suppressed with dithiothreitol (DTT = 1 mM) (n = 0/2/group) or RLX (low or high dose, n = 0/6/group). PAH hearts developed increased fibrosis that was reversed by RLX-HD, but not RLX-LD. Relaxin decreased Nrf2 and glutathione transferases but not glutathione-reductase. High-dose RLX improved pulmonary arterial compliance (measured by Doppler flow), suppressed VF even after burst-pacing, n = 2/6). Relaxin suppressed VF and asystole through electrical remodeling and by reversing thiol oxidative stress. For the first time, we showed two cardiac phenotypes in PAH animals and their prevention by RLX. Relaxin may modulate maladaptive cardiac remodeling in PAH and protect against arrhythmia and cardiac arrest.

Relaxin abrogates genomic remodeling of the aged heart.

"Healthy" aging drives structural and functional changes in the heart including maladaptive electrical remodeling, fibrosis and inflammation, which lower the threshold for cardiovascular diseases such as heart failure (HF) and atrial fibrillation (AF). Despite mixed results in clinical trials, Relaxin-therapy for 2-days reduced mortality by 37% at 180-days post-treatment, in patients with acute decompensated HF. Relaxin's short lifespan (2-3h) but long-lasting protective actions suggested that relaxin acts at a genomic level to reverse maladaptive remodeling in AF, HF and aging. Our recent studies showed that a 2-week treatment with Relaxin (0.4mg/kg/day) of aged (24months old F-344 rats) increases the expression of voltage-gated Na+ channels (mRNA, Nav1.5 and INa), connexin-43, abrogates inflammatory and immune responses and reverses myocardial fibrosis and cellular hypertrophy of the aged hearts. Relaxin acts directly at a wide range of cell types in the cardiovascular system that express its cognate GPCR receptor, RXFP1. RNA-seq analysis of young and aged hearts with and without Relaxin treatment revealed that "normal" aging altered the expression of ~10% of genes expressed in the ventricles, including: ion channels, components of fibrosis, hemodynamic biomarkers, immune and inflammatory responses which were reversed by Relaxin. The extensive cardiovascular remodeling caused by Relaxin was mediated through the activation of the Wnt/ß-catenin signaling pathway which was otherwise suppressed by in adult cardiomyocytes intracellular by cytosolic Dickkopf1 (Dkk1). Wnt/ß-catenin signaling is a mechanism that can explain the pleiotropic actions of Relaxin and the marked reversal of genomic changes that occur in aged hearts.

Relaxin reverses maladaptive remodeling of the aged heart through Wnt-signaling.

Healthy aging results in cardiac structural and electrical remodeling that increases susceptibility to cardiovascular diseases. Relaxin, an insulin-like hormone, suppresses atrial fibrillation, inflammation and fibrosis in aged rats but the mechanisms-of-action are unknown. Here we show that relaxin treatment of aged rats reverses pathological electrical remodeling (increasing Nav1.5 expression and localization of Connexin43 to intercalated disks) by activating canonical Wnt signaling. In isolated adult ventricular myocytes, relaxin upregulated Nav1.5 (EC50 = 1.3 nM) by a mechanism inhibited by the addition of Dickkopf-1. Furthermore, relaxin increased the levels of connexin43, Wnt1, and cytosolic and nuclear ß-catenin. Treatment with Wnt1 or CHIR-99021 (a GSK3ß inhibitor) mimicked the relaxin effects. In isolated fibroblasts, relaxin blocked TGFß-induced collagen elevation in a Wnt dependent manner. These findings demonstrate a close interplay between relaxin and Wnt-signaling resulting in myocardial remodeling and reveals a fundamental mechanism of great therapeutic potential.

Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies.

Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs.

Cardioprotective actions of relaxin.

Relaxin is a hormone of pregnancy first discovered for its ability to induce ligament relaxation in nonpregnant guinea pig and is important for softening of the birth canal during parturition, decidualization, implantation, nipple development and increased maternal renal perfusion, glomerular filtration, and cardiac output. Subsequently, relaxin has been shown to exert multiple beneficial cardiovascular effects during pathological events such as hypertension, atrial fibrillation, heart failure and myocardial infarction, including suppression of arrhythmia and inflammation, and reversal of fibrosis. Despite extensive studies, the mechanisms underlying relaxin's effects are not well understood. Relaxin signals primarily through its G protein coupled receptor, the relaxin family peptide receptor-1, to activate multiple signaling pathways and this review summarizes our understanding of these pathways as they relate to the cardioprotective actions of relaxin, focusing on relaxin's anti-fibrotic, anti-arrhythmic and anti-inflammatory properties. Further, this review includes a brief overview of relaxin in clinical trials for heart failure and progress in the development of relaxin mimetics.

Overexpression of Cx43 in cells of the myocardial scar: Correction of post-infarct arrhythmias through heterotypic cell-cell coupling.

Ventricular tachycardia (VT) is the most common and potentially lethal complication following myocardial infarction (MI). Biological correction of the conduction inhomogeneity that underlies re-entry could be a major advance in infarction therapy. As minimal increases in conduction of infarcted tissue markedly influence VT susceptibility, we reasoned that enhanced propagation of the electrical signal between non-excitable cells within a resolving infarct might comprise a simple means to decrease post-infarction arrhythmia risk. We therefore tested lentivirus-mediated delivery of the gap-junction protein Connexin 43 (Cx43) into acute myocardial lesions. Cx43 was expressed in (myo)fibroblasts and CD45+ cells within the scar and provided prominent and long lasting arrhythmia protection in vivo. Optical mapping of Cx43 injected hearts revealed enhanced conduction velocity within the scar, indicating Cx43-mediated electrical coupling between myocytes and (myo)fibroblasts. Thus, Cx43 gene therapy, by direct in vivo transduction of non-cardiomyocytes, comprises a simple and clinically applicable biological therapy that markedly reduces post-infarction VT.

Estradiol up-regulates L-type Ca2+ channels via membrane-bound estrogen receptor/phosphoinositide-3-kinase/Akt/cAMP response element-binding protein signaling pathway.

BACKGROUND: In long QT syndrome type 2, women are more prone than men to the lethal arrhythmia torsades de pointes. We previously reported that 17ß-estradiol (E2) up-regulates L-type Ca2+ channels and current (ICa,L) (∼30%) in rabbit ventricular myocytes by a classic genomic mechanism mediated by estrogen receptor-α (ERα). In long QT syndrome type 2 (IKr blockade or bradycardia), the higher Ca2+ influx via ICa,L causes Ca2+ overload, spontaneous sarcoplasmic reticulum Ca2+ release, and reactivation of ICa,L that triggers early afterdepolarizations and torsades de pointes. OBJECTIVE: The purpose of this study was to investigate the molecular mechanisms whereby E2 up-regulates ICa,L, which are poorly understood. METHODS: H9C2 and rat myocytes were incubated with E2 ± ER antagonist, or inhibitors of downstream transcription factors, for 24 hours, followed by western blots of Cav1.2α1C and voltage-clamp measurements of ICa,L. RESULTS: Incubation of H9C2 cells with E2 (10-100 nM) increased ICa,L density and Cav1.2α1C expression, which were suppressed by the ER antagonist ICI182,780 (1 µM). Enhanced ICa,L and Cav1.2α1C expression by E2 was suppressed by inhibitors of phosphoinositide-3-kinase (Pi3K) (30 µM LY294002; P <.05) and Akt (5 µM MK2206) but not of mitogen-activated protein kinase (5 µM U0126) or protein kinase A (1 µM KT5720). E2 incubation increased p-CREB via the Pi3K/Akt pathway, reached a peak in 20 minutes (3-fold), and leveled off to 1.5-fold 24 hours later. Furthermore, a CREB decoy oligonucleotide inhibited E2-induced Cav1.2α1C expression, whereas membrane-impermeable E2 (E2-bovine serum albumin) was equally effective at Cav1.2α1C up-regulation as E2. CONCLUSION: Estradiol up-regulates Cav1.2α1C and ICa,L via plasma membrane ER and by activating Pi3K, Akt, and CREB signaling. The promoter regions of the CACNA1C gene (human-rabbit-rat) contain adjacent/overlapping binding sites for p-CREB and ERα, which suggests a synergistic regulation by these pathways.

Relaxin reverses inflammatory and immune signals in aged hearts.

BACKGROUND: 'Healthy' aging drives structural and functional changes in the heart including maladaptive electrical remodeling, fibrosis and inflammation, which lower the threshold for cardiovascular diseases such as heart failure (HF) and atrial fibrillation (AF). Despite mixed results in recent clinical trials, Relaxin-therapy for 2-days could reduce mortality by 37% at 180-days post-treatment, in patients with acute decompensated HF. Relaxin's short life-span (hours) but long-lasting protective actions led us to test the hypothesis that relaxin acts at a genomic level to reverse maladaptive remodeling in aging and HF. METHODS AND RESULTS: Young (9-month) and aged (24-month), male and female F-344/Brown Norway rats were treated with relaxin (0.4 mg/kg/day) for 2-weeks delivered by subcutaneous osmotic mini-pumps or with sodium acetate (controls). The genomic effects of aging and relaxin were evaluated by extracting RNA from the left ventricles and analyzing genomic changes by RNA-sequencing, Ingenuity Pathway Analysis, MetaCore and tissue immunohistochemistry. We found that aging promotes a native inflammatory response with distinct sex-differences and relaxin suppresses transcription of multiple genes and signaling pathways associated with inflammation and HF in both genders. In addition, aging significantly increased: macrophage infiltration and atrial natriuretic peptide levels in female ventricles, and activation of the complement cascade, whereas relaxin reversed these age-related effects. CONCLUSION: These data support the hypothesis that relaxin alters gene transcription and suppresses inflammatory pathways and genes associated with HF and aging. Relaxin's suppression of inflammation and fibrosis supports its potential as a therapy for cardiovascular and inflammation-related diseases, such as HF, AF and diabetes.