The Longitudinal Decline in Peak VO2 with Aging in Healthy Individuals is Associated with a Reduction in Peripheral Oxygen Utilization but not in Cardiac Output.

Aging is associated with a significant decline in exercise fitness assessed by maximal exercise oxygen consumption (VO2-max). The specific VO2-max components driving this decline, namely cardiac output (CO) and arteriovenous oxygen difference (A-V) O2, remain unclear. We examined this issue by analyzing data from 99 community-dwelling participants (baseline age 21-96 years; average follow-up 12.6 years) from the Baltimore Longitudinal Study of Aging, free of clinical cardiovascular disease. VO2-peak, a surrogate of VO2-max, was used to assess aerobic capacity during upright cycle exercise. Peak exercise left ventricular (LV) volumes, heart rate, and cardiac output were estimated using repeated gated cardiac blood pool scans. The Fick equation was used to calculate (A-V) O2-peak from CO-peak and VO2-peak. In unadjusted models, VO2-peak, (A-V) O2-peak, and CO-peakdeclined longitudinally over time at steady rates with advancing age. In multiple linear regression models adjusting for baseline values and peak workload, however, steeper declines in VO2-peak and (A-V) O2 peak were observed with advanced entry age but not in CO-peak. The association between the declines in VO2-peak and (A-V) O2-peakwas stronger among those >=50 years compared to their younger counterparts but the difference between the two age groups did not reach statistical significance. These findings suggest that age-associated impairment of peripheral oxygen utilization during maximal exercise poses a stronger limitation on peak VO2 than that of CO. Future studies examining interventions targeting the structure and function of peripheral muscles and their vasculature to mitigate age-associated declines in (A-V) O2 are warranted.

Transcriptional regulation of intermolecular Ca2+ signaling in hibernating ground squirrel cardiomyocytes: The myocardin-junctophilin axis.

The contraction of heart cells is controlled by the intermolecular signaling between L-type Ca2+ channels (LCCs) and ryanodine receptors (RyRs), and the nanodistance between them depends on the interaction between junctophilin-2 (JPH2) in the sarcoplasmic reticulum (SR) and caveolin-3 (CAV3) in the transversal tubule (TT). In heart failure, decreased expression of JPH2 compromises LCC-RyR communication leading to deficient blood-pumping power. In the present study, we found that JPH2 and CAV3 transcription was concurrently regulated by serum response factor (SRF) and myocardin. In cardiomyocytes from torpid ground squirrels, compared with those from euthermic counterparts, myocardin expression was up-regulated, which boosted both JPH2 and CAV3 expression. Transmission electron microscopic imaging showed that the physical coupling between TTs and SRs was tightened during hibernation and after myocardin overexpression. Confocal Ca2+ imaging under the whole-cell patch clamp condition revealed that these changes enhanced the efficiency of LCC-RyR intermolecular signaling and fully compensated the adaptive down-regulation of LCCs, maintaining the power of heart contraction while avoiding the risk of calcium overload during hibernation. Our finding not only revealed an essential molecular mechanism underlying the survival of hibernating mammals, but also demonstrated a "reverse model of heart failure" at the molecular level, suggesting a strategy for treating heart diseases.

Superoxide flashes in single mitochondria.

In quiescent cells, mitochondria are the primary source of reactive oxygen species (ROS), which are generated by leakiness of the electron transport chain (ETC). High levels of ROS can trigger cell death, whereas lower levels drive diverse and important cellular functions. We show here by employing a newly developed mitochondrial matrix-targeted superoxide indicator, that individual mitochondria undergo spontaneous bursts of superoxide generation, termed "superoxide flashes." Superoxide flashes occur randomly in space and time, exhibit all-or-none properties, and provide a vital source of superoxide production across many different cell types. Individual flashes are triggered by transient openings of the mitochondrial permeability transition pore stimulating superoxide production by the ETC. Furthermore, we observe a flurry of superoxide flash activity during reoxygenation of cardiomyocytes after hypoxia, which is inhibited by the cardioprotective compound adenosine. We propose that superoxide flashes could serve as a valuable biomarker for a wide variety of oxidative stress-related diseases.

Photobiomodulation therapy mitigates cardiovascular aging and improves survival.

BACKGROUND: Photobiomodulation (PBM) therapy, a form of low-dose light therapy, has been noted to be effective in several age-associated chronic diseases such as hypertension and atherosclerosis. Here, we examined the effects of PBM therapy on age-associated cardiovascular changes in a mouse model of accelerated cardiac aging. METHODS: Fourteen months old Adenylyl cyclase type VIII (AC8) overexpressing transgenic mice (n = 8) and their wild-type (WT) littermates (n = 8) were treated with daily exposure to Near-Infrared Light (850 nm) at 25 mW/cm2 for 2 min each weekday for a total dose of 1 Einstein (4.5 p.J/cm2 or fluence 3 J/cm2 ) and compared to untreated controls over an 8-month period. PBM therapy was administered for 3.5 months (Early Treatment period), paused, due to Covid-19 restrictions for the following 3 months, and restarted again for 1.5 months. Serial echocardiography and gait analyses were performed at monthly intervals, and serum TGF-ß1 levels were assessed following sacrifice. RESULTS: During the Early Treatment period PBM treatments: reduced the age-associated increases in left ventricular (LV) mass in both genotypes (p = 0.0003), reduced the LV end-diastolic volume (EDV) in AC8 (p = 0.04); and reduced the left atrial dimension in both genotypes (p = 0.02). PBM treatments substantially increased the LV ejection fraction (p = 0.03), reduced the aortic wall stiffness (p = 0.001), and improved gait symmetry, an index of neuro-muscular coordination (p = 0.005). The effects of PBM treatments, measured following the pause, persisted. Total TGF-ß1 levels were significantly increased in circulation (serum) in AC8 following PBM treatments (p = 0.01). We observed a striking increase in cumulative survival in PBM-treated AC8 mice (100%; p = 0.01) compared to untreated AC8 mice (43%). CONCLUSION: PBM treatment mitigated age-associated cardiovascular remodeling and reduced cardiac function, improved neuromuscular coordination, and increased longevity in an experimental animal model. These responses correlate with increased TGF-ß1 in circulation. Future mechanistic and dose optimization studies are necessary to assess these anti-aging effects of PBM, and validation in future controlled human studies is required for effective clinical translation.

Increased Drp1 Acetylation by Lipid Overload Induces Cardiomyocyte Death and Heart Dysfunction.

RATIONALE: Lipid overload-induced heart dysfunction is characterized by cardiomyocyte death, myocardial remodeling, and compromised contractility, but the impact of excessive lipid supply on cardiac function remains poorly understood. OBJECTIVE: To investigate the regulation and function of the mitochondrial fission protein Drp1 (dynamin-related protein 1) in lipid overload-induced cardiomyocyte death and heart dysfunction. METHODS AND RESULTS: Mice fed a high-fat diet (HFD) developed signs of obesity and type II diabetes mellitus, including hyperlipidemia, hyperglycemia, hyperinsulinemia, and hypertension. HFD for 18 weeks also induced heart hypertrophy, fibrosis, myocardial insulin resistance, and cardiomyocyte death. HFD stimulated mitochondrial fission in mouse hearts. Furthermore, HFD increased the protein level, phosphorylation (at the activating serine 616 sites), oligomerization, mitochondrial translocation, and GTPase activity of Drp1 in mouse hearts, indicating that Drp1 was activated. Monkeys fed a diet high in fat and cholesterol for 2.5 years also exhibited myocardial damage and Drp1 activation in the heart. Interestingly, HFD decreased nicotinamide adenine dinucleotide (oxidized) levels and increased Drp1 acetylation in the heart. In adult cardiomyocytes, palmitate increased Drp1 acetylation, phosphorylation, and protein levels, and these increases were abolished by restoration of the decreased nicotinamide adenine dinucleotide (oxidized) level. Proteomics analysis and in vitro screening revealed that Drp1 acetylation at lysine 642 (K642) was increased by HFD in mouse hearts and by palmitate incubation in cardiomyocytes. The nonacetylated Drp1 mutation (K642R) attenuated palmitate-induced Drp1 activation, its interaction with voltage-dependent anion channel 1, mitochondrial fission, contractile dysfunction, and cardiomyocyte death. CONCLUSIONS: These findings uncover a novel mechanism that contributes to lipid overload-induced heart hypertrophy and dysfunction. Excessive lipid supply created an intracellular environment that facilitated Drp1 acetylation, which, in turn, increased its activity and mitochondrial translocation, resulting in cardiomyocyte dysfunction and death. Thus, Drp1 may be a critical mediator of lipid overload-induced heart dysfunction as well as a potential target for therapy.

Metformin Attenuates Hyperglycaemia-Stimulated Pro-Fibrotic Gene Expression in Adventitial Fibroblasts via Inhibition of Discoidin Domain Receptor 2.

Molecular mechanisms underlying the diverse therapeutic effects of anti-diabetic metformin, beyond its anti-hyperglycaemic effects, remain largely unclear. Metformin is reported to reduce the long-term complications of diabetes, including cardiovascular fibrosis and remodelling. Our recent investigations show that Discoidin Domain Receptor 2 (DDR2), a Collagen receptor tyrosine kinase, has an obligate regulatory role in Collagen type I gene expression in cardiac and vascular adventitial fibroblasts, and that it may be a molecular link between arterial fibrosis and metabolic syndrome in rhesus monkeys. Using gene knockdown and overexpression approaches, the present study examined whether DDR2 is a target of metformin and whether, by targeting DDR2, it inhibits Fibronectin and Collagen type I expression in rat aortic adventitial fibroblasts exposed to hyperglycaemic conditions. Metformin was found to attenuate hyperglycaemia-induced increase in DDR2 mRNA and protein expression by inhibiting TGF-ß1/SMAD2/3 signalling that mediates the stimulatory effect of hyperglycaemia on DDR2 expression. Metformin also inhibited DDR2-dependent expression of Fibronectin and Collagen type I, indicating that it regulates these matrix proteins via DDR2 inhibition. The findings identify DDR2, a mediator of cardiovascular remodelling, as a molecular target of metformin, thereby uncovering the molecular basis of its protective role in vascular fibrosis and possibly cardiac fibrosis associated with diabetic cardiomyopathy.

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.

Dual Activation of Phosphodiesterase 3 and 4 Regulates Basal Cardiac Pacemaker Function and Beyond.

The sinoatrial (SA) node is the physiological pacemaker of the heart, and resting heart rate in humans is a well-known risk factor for cardiovascular disease and mortality. Consequently, the mechanisms of initiating and regulating the normal spontaneous SA node beating rate are of vital importance. Spontaneous firing of the SA node is generated within sinoatrial nodal cells (SANC), which is regulated by the coupled-clock pacemaker system. Normal spontaneous beating of SANC is driven by a high level of cAMP-mediated PKA-dependent protein phosphorylation, which rely on the balance between high basal cAMP production by adenylyl cyclases and high basal cAMP degradation by cyclic nucleotide phosphodiesterases (PDEs). This diverse class of enzymes includes 11 families and PDE3 and PDE4 families dominate in both the SA node and cardiac myocardium, degrading cAMP and, consequently, regulating basal cardiac pacemaker function and excitation-contraction coupling. In this review, we will demonstrate similarities between expression, distribution, and colocalization of various PDE subtypes in SANC and cardiac myocytes of different species, including humans, focusing on PDE3 and PDE4. Here, we will describe specific targets of the coupled-clock pacemaker system modulated by dual PDE3 + PDE4 activation and provide evidence that concurrent activation of PDE3 + PDE4, operating in a synergistic manner, regulates the basal cardiac pacemaker function and provides control over normal spontaneous beating of SANCs through (PDE3 + PDE4)-dependent modulation of local subsarcolemmal Ca2+ releases (LCRs).

Discoidin Domain Receptor 2 Regulates AT1R Expression in Angiotensin II-Stimulated Cardiac Fibroblasts via Fibronectin-Dependent Integrin-ß1 Signaling.

This study probed the largely unexplored regulation and role of fibronectin in Angiotensin II-stimulated cardiac fibroblasts. Using gene knockdown and overexpression approaches, Western blotting, and promoter pull-down assay, we show that collagen type I-activated Discoidin Domain Receptor 2 (DDR2) mediates Angiotensin II-dependent transcriptional upregulation of fibronectin by Yes-activated Protein in cardiac fibroblasts. Furthermore, siRNA-mediated fibronectin knockdown attenuated Angiotensin II-stimulated expression of collagen type I and anti-apoptotic cIAP2, and enhanced cardiac fibroblast susceptibility to apoptosis. Importantly, an obligate role for fibronectin was observed in Angiotensin II-stimulated expression of AT1R, the Angiotensin II receptor, which would link extracellular matrix (ECM) signaling and Angiotensin II signaling in cardiac fibroblasts. The role of fibronectin in Angiotensin II-stimulated cIAP2, collagen type I, and AT1R expression was mediated by Integrin-ß1-integrin-linked kinase signaling. In vivo, we observed modestly reduced basal levels of AT1R in DDR2-null mouse myocardium, which were associated with the previously reported reduction in myocardial Integrin-ß1 levels. The role of fibronectin, downstream of DDR2, could be a critical determinant of cardiac fibroblast-mediated wound healing following myocardial injury. In summary, our findings suggest a complex mechanism of regulation of cardiac fibroblast function involving two major ECM proteins, collagen type I and fibronectin, and their receptors, DDR2 and Integrin-ß1.

Cardiotonic Steroids Induce Vascular Fibrosis Via Pressure-Independent Mechanism in NaCl-Loaded Diabetic Rats.

Endogenous cardiotonic steroid, marinobufagenin (MBG), induces Fli1-dependent tissue fibrosis. We hypothesized that an increase in MBG initiates the development of aortic fibrosis in salt-loaded rats with type 2 diabetes mellitus (DM2) via pressure-independent mechanism. DM2 was induced by a single intraperitoneal administration of 65 mg/kg streptozotocin to neonatal (4-5 days) male Wistar rats. Eight-week-old DM2 rats received water or 1.8% NaCl (DM-NaCl) solution for 4 weeks (n = 16); half of DM-NaCl rats were treated with anti-MBG monoclonal antibody (mAb) (DM-NaCl-AB) during week 4 of salt loading; control intact rats received water (n = 8/group). Blood pressure, MBG, erythrocyte Na/K-ATPase activity, aortic weights, levels of fibrosis markers (Fli1, protein kinase Cδ, transforming growth factor-ß1, receptors of the transforming growth factor beta5, fibronectin, collagen-1), and sensitivity of the aortic explants to the vasorelaxant effect of sodium nitroprusside were assessed. No changes in systolic blood pressure were observed while erythrocyte Na/K-ATPase was inhibited by 30%, plasma MBG was doubled, and aortic markers of fibrosis became elevated in DM-NaCl rats versus control. Treatment of DM-NaCl rats with anti-MBG mAb activated Na/K-ATPase, prevented increases in aortic weights, and the levels of fibrosis markers returned to the control levels. The responsiveness of the aortic rings from DM-NaCl rats to the relaxant effect of sodium nitroprusside was reduced (half maximal effective concentration (EC50) = 29 nmol/L) versus control rings (EC50 = 7 nmol/L) and was restored by anti-MBG mAb (EC50 = 9 nmol/L). Our results suggest that in salt-loaded diabetic rats, MBG stimulates aortic collagen synthesis in a pressure-independent fashion and that 2 profibrotic mechanisms, Fli1 dependent and transforming growth factor-ß dependent, underlie its effects.

Acute salt loading and cardiotonic steroids in resistant hypertension.

The study addresses the association of marinobufagenin (MBG), a natriuretic and vasoconstrictor steroid, and Na/K-ATPase (NKA) activity with pressor response to salt-loading and arterial stiffness in resistant hypertension (RH). Thirty-four patients (18 males and 16 females; 56±8 years) with RH on a combined (lisnopril/amlodipine/hydrochlorothiazide) therapy and 11 healthy age-matched normotensive subjects (7 males and 4 females; 54±2 years) were enrolled in this study. Salt-loading was performed via intravenous infusion of 1000mL saline (0.9% NaCl) for 1h. Arterial stiffness was measured by Sphygmocor Px device with a calculation of pulse-wave velocity (PWV). Activity of NKA was measured in erythrocytes. We demonstrated that plasma levels of MBG and magnitude of NaCl-induced MBG-dependent NKA inhibition are associated with PWV, and that this association has gender- and age-specific fashion in RH patients.

Positive Feedback Mechanisms among Local Ca Releases, NCX, and ICaL Ignite Pacemaker Action Potentials.

Recent data suggest that cardiac pacemaker cell function is determined by numerous time-, voltage-, and Ca-dependent interactions of cell membrane electrogenic proteins (M-clock) and intracellular Ca cycling proteins (Ca-clock), forming a coupled-clock system. Many aspects of the coupled-clock system, however, remain underexplored. The key players of the system are Ca release channels (ryanodine receptors), generating local Ca releases (LCRs) from sarcoplasmic reticulum, electrogenic Na/Ca exchanger (NCX) current, and L-type Ca current (ICaL). We combined numerical model simulations with experimental simultaneous recordings of action potentials (APs) and Ca to gain further insight into the complex interactions within the system. Our simulations revealed a positive feedback mechanism, dubbed AP ignition, which accelerates the diastolic depolarization (DD) to reach AP threshold. The ignition phase begins when LCRs begin to occur and the magnitude of inward NCX current begins to increase. The NCX current, together with funny current and T-type Ca current accelerates DD, bringing the membrane potential to ICaL activation threshold. During the ignition phase, ICaL-mediated Ca influx generates more LCRs via Ca-induced Ca release that further activates inward NCX current, creating a positive feedback. Simultaneous recordings of membrane potential and confocal Ca images support the model prediction of the positive feedback among LCRs and ICaL, as diastolic LCRs begin to occur below and continue within the voltage range of ICaL activation. The ignition phase onset (identified within the fine DD structure) begins when DD starts to notably accelerate (∼0.15 V/s) above the recording noise. Moreover, the timing of the ignition onset closely predicted the duration of each AP cycle in the basal state, in the presence of autonomic receptor stimulation, and in response to specific inhibition of either the M-clock or Ca-clock, thus indicating general importance of the new coupling mechanism for regulation of the pacemaker cell cycle duration, and ultimately the heart rate.

Electrophysiological heterogeneity of pacemaker cells in the rabbit intercaval region, including the SA node: insights from recording multiple ion currents in each cell.

Cardiac pacemaker cells, including cells of the sinoatrial node, are heterogeneous in size, morphology, and electrophysiological characteristics. The exact extent to which these cells differ electrophysiologically is unclear yet is critical to understanding their functioning. We examined major ionic currents in individual intercaval pacemaker cells (IPCs) sampled from the paracristal, intercaval region (including the sinoatrial node) that were spontaneously beating after enzymatic isolation from rabbit hearts. The beating rate was measured at baseline and after inhibition of the Ca2+ pump with cyclopiazonic acid. Thereafter, in each cell, we consecutively measured the density of funny current ( If), delayed rectifier K+ current ( IK) (a surrogate of repolarization capacity), and L-type Ca2+ current ( ICa,L) using whole cell patch clamp. The ionic current densities varied to a greater extent than previously appreciated, with some IPCs demonstrating very small or zero If . The density of none of the currents was correlated with cell size, while ICa,L and If densities were related to baseline beating rates. If density was correlated with IK density but not with that of ICa,L. Inhibition of Ca2+ cycling had a greater beating rate slowing effect in IPCs with lower If densities. Our numerical model simulation indicated that 1) IPCs with small (or zero) If or small ICa,L can operate via a major contribution of Ca2+ clock, 2) If-Ca2+-clock interplay could be important for robust pacemaking function, and 3) coupled If- IK function could regulate maximum diastolic potential. Thus, we have demonstrated marked electrophysiological heterogeneity of IPCs. This heterogeneity is manifested in basal beating rate and response to interference of Ca2+ cycling, which is linked to If. NEW & NOTEWORTHY In the present study, a hitherto unrecognized range of heterogeneity of ion currents in pacemaker cells from the intercaval region is demonstrated. Relationships between basal beating rate and L-type Ca2+ current and funny current ( If) density are uncovered, along with a positive relationship between If and delayed rectifier K+ current. Links are shown between the response to Ca2+ cycling blockade and If density.

Adipocytokine correlations with thyroid function and autoimmunity in euthyroid sardinians.

OBJECTIVE: Cytokines release by adipocytes could interact with TSH secretion. We evaluated the relationship between adipocytokines and TSH. We further tested for association of cytokines and thyroid autoimmunity. METHODS: We conducted a cross-sectional study in a community-based sample including 5385 individuals (2964 female) with TSH within the reference range. Subjects who reported taking thyroid medications or drugs that alter thyroid function were excluded. TSH, FT4, adiponectin, leptin, antibody against thyroperoxidase and against thyroglobulin were measured. Linear and logistic regression models were used to test for association. RESULTS: Females had higher adiponectin and leptin level and increased frequency of thyroid antibodies. In multiple regression analysis TSH was directly associated with leptin (ß = 0.003, p = 0.001) and the presence of circulating antibody against thyroperoxidase (ß = 0.315, p < 0.001), but negatively associated with age (ß = -0.012, p < 0.001) and FT4 (ß = -0.359, p < 0.001). Adiponectin, the presence of antibody against thyroglobulin and smoking habit were not associated with TSH levels (p = 0.223, p = 0.174 and p = 0.788, respectively). Logistic regression analysis revealed that higher adiponectin levels were associated with thyroid autoimmunity. CONCLUSIONS: Leptin is positively associated with TSH levels in euthyroid individuals, suggesting an effect of the adipokine on TSH secretion. Our results support the hypothesis that the leptin and pituitary-thyroid axis might interact in the context of energy homeostasis. The effect of adiponectin on thyroid autoimmunity will require more studies.

Pharmacological Strategies to Retard Cardiovascular Aging.

Aging is the major risk factor for cardiovascular diseases, which are the leading cause of death in the United States. Traditionally, the effort to prevent cardiovascular disease has been focused on addressing the conventional risk factors, including hypertension, hyperglycemia, hypercholesterolemia, and high circulating levels of triglycerides. However, recent preclinical studies have identified new approaches to combat cardiovascular disease. Calorie restriction has been reproducibly shown to prolong lifespan in various experimental model animals. This has led to the development of calorie restriction mimetics and other pharmacological interventions capable to delay age-related diseases. In this review, we will address the mechanistic effects of aging per se on the cardiovascular system and focus on the prolongevity benefits of various therapeutic strategies that support cardiovascular health.

Unique Ca2+-Cycling Protein Abundance and Regulation Sustains Local Ca2+ Releases and Spontaneous Firing of Rabbit Sinoatrial Node Cells.

Spontaneous beating of the heart pacemaker, the sinoatrial node, is generated by sinoatrial node cells (SANC) and caused by gradual change of the membrane potential called diastolic depolarization (DD). Submembrane local Ca2+ releases (LCR) from sarcoplasmic reticulum (SR) occur during late DD and activate an inward Na⁺/Ca2+ exchange current, which accelerates the DD rate leading to earlier occurrence of an action potential. A comparison of intrinsic SR Ca2+ cycling revealed that, at similar physiological Ca2+ concentrations, LCRs are large and rhythmic in permeabilized SANC, but small and random in permeabilized ventricular myocytes (VM). Permeabilized SANC spontaneously released more Ca2+ from SR than VM, despite comparable SR Ca2+ content in both cell types. In this review we discuss specific patterns of expression and distribution of SR Ca2+ cycling proteins (SR Ca2+ ATPase (SERCA2), phospholamban (PLB) and ryanodine receptors (RyR)) in SANC and ventricular myocytes. We link ability of SANC to generate larger and rhythmic LCRs with increased abundance of SERCA2, reduced abundance of the SERCA inhibitor PLB. In addition, an increase in intracellular [Ca2+] increases phosphorylation of both PLB and RyR exclusively in SANC. The differences in SR Ca2+ cycling protein expression between SANC and VM provide insights into diverse regulation of intrinsic SR Ca2+ cycling that drives automaticity of SANC.

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.

Antibody to Marinobufagenin Reverses Placenta-Induced Fibrosis of Umbilical Arteries in Preeclampsia.

BACKGROUND: Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Immunoneutralization of heightened MBG by Digibind, a digoxin antibody, reduces blood pressure (BP) in patients with PE, and anti-MBG monoclonal antibody lessens BP in a rat model of PE. Recently, we demonstrated that MBG induces fibrosis in cardiovascular tissues via a mechanism involving inhibition of Fli-1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis. OBJECTIVES AND METHODS: We hypothesized that in PE, elevated placental MBG levels are associated with development of fibrosis in umbilical arteries. Eleven patients with PE (mean BP 124 ± 4 mmHg; age 29 ± 2 years; 39 weeks gest. age) and 10 gestational age-matched normal pregnant subjects (mean BP 92 ± 2 mmHg; controls) were enrolled in the clinical study. RESULTS: PE was associated with a higher placental (0.04 ± 0.01 vs. 0.49 ± 0.11 pmol/g; p < 0.01) and plasma MBG (0.5 ± 0.1 vs. 1.6 ± 0.5 nmol/L; p < 0.01), lower Na/K-ATPase activity in erythrocytes (2.7 ± 0.2 vs. 1.5 ± 0.2 µmol Pi/mL/hr; p < 0.01), 9-fold decrease of Fli-1 level and 2.5-fold increase of collagen-1 in placentae (p < 0.01) vs. control. Incubation of umbilical arteries from control patients with 1 nmol/L MBG was associated with four-fold decrease in Fli-1 level and two-fold increase in collagen-1 level vs. those incubated with placebo (p < 0.01), i.e., physiological concentration of MBG mimicked effect of PE in vitro. Collagen-1 abundance in umbilical arteries from PE patients was 4-fold higher than in control arteries, and this PE-associated fibrosis was reversed by monoclonal anti-MBG antibody ex vivo. CONCLUSION: These results demonstrate that elevated placental MBG level is implicated in the development of fibrosis of the placenta and umbilical arteries in PE.

Long-term low dose dietary resveratrol supplement reduces cardiovascular structural and functional deterioration in chronic heart failure in rats.

A short-term exposure to resveratrol at high dosages exerts a remarkable cardioprotective effect. Whether a long-term exposure to resveratrol at low dosages that can be obtained through consumption of a resveratrol-rich diet is beneficial to heart diseases is unknown. We tested the effects of a resveratrol-enriched diet on cardiovascular remodeling of chronic heart failure (CHF) in rats resulting from permanent ligation of left coronary artery. Two weeks after surgery, rats were started on either a resveratrol-enriched (R; 5 mg/kg per day; n = 23) or normal (Control; n = 23) diet for next 10 months. Serial echocardiography in Control showed a significant decline in LV ejection fraction, increases in LV end-systolic and end-diastolic volumes, and expansion in myocardial infarct from pre-treatment values. In R, compared with Control, there were substantial improvements in those parameters. End-point LV pressure-volume loop analysis showed a significantly improved LV systolic function and AV-coupling, an index of energy transfer efficacy between the heart and aortic tree, in R compared with Control (p < 0.05). Aortic pulse wave velocity, a measure of arterial stiffness, was significantly lower in R (389 ± 15 cm/s; p < 0.05) compared with Control (489 ± 38 cm/s). These results demonstrated that long-term dietary resveratrol supplement reduces cardiovascular structural and functional deterioration in CHF.

Ca(2+)/calmodulin-activated phosphodiesterase 1A is highly expressed in rabbit cardiac sinoatrial nodal cells and regulates pacemaker function.

Constitutive Ca(2+)/calmodulin (CaM)-activation of adenylyl cyclases (ACs) types 1 and 8 in sinoatrial nodal cells (SANC) generates cAMP within lipid-raft-rich microdomains to initiate cAMP-protein kinase A (PKA) signaling, that regulates basal state rhythmic action potential firing of these cells. Mounting evidence in other cell types points to a balance between Ca(2+)-activated counteracting enzymes, ACs and phosphodiesterases (PDEs) within these cells. We hypothesized that the expression and activity of Ca(2+)/CaM-activated PDE Type 1A is higher in SANC than in other cardiac cell types. We found that PDE1A protein expression was 5-fold higher in sinoatrial nodal tissue than in left ventricle, and its mRNA expression was 12-fold greater in the corresponding isolated cells. PDE1 activity (nimodipine-sensitive) accounted for 39% of the total PDE activity in SANC lysates, compared to only 4% in left ventricular cardiomyocytes (LVC). Additionally, total PDE activity in SANC lysates was lowest (10%) in lipid-raft-rich and highest (76%) in lipid-raft-poor fractions (equilibrium sedimentation on a sucrose density gradient). In intact cells PDE1A immunolabeling was not localized to the cell surface membrane (structured illumination microscopy imaging), but located approximately within about 150nm inside of immunolabeling of hyperpolarization-activated cyclic nucleotide-gated potassium channels (HCN4), which reside within lipid-raft-rich microenvironments. In permeabilized SANC, in which surface membrane ion channels are not functional, nimodipine increased spontaneous SR Ca(2+) cycling. PDE1A mRNA silencing in HL-1 cells increased the spontaneous beating rate, reduced the cAMP, and increased cGMP levels in response to IBMX, a broad spectrum PDE inhibitor (detected via fluorescence resonance energy transfer microscopy). We conclude that signaling via cAMP generated by Ca(2+)/CaM-activated AC in SANC lipid raft domains is limited by cAMP degradation by Ca(2+)/CaM-activated PDE1A in non-lipid raft domains. This suggests that local gradients of [Ca(2+)]-CaM or different AC and PDE1A affinity regulate both cAMP production and its degradation, and this balance determines the intensity of Ca(2+)-AC-cAMP-PKA signaling that drives SANC pacemaker function.