Extracellular Matrix Disarray as a Mechanism for Greater Abdominal Versus Thoracic Aortic Stiffness With Aging in Primates.

OBJECTIVE: Increased vascular stiffness is central to the pathophysiology of aging, hypertension, diabetes mellitus, and atherosclerosis. However, relatively few studies have examined vascular stiffness in both the thoracic and the abdominal aorta with aging, despite major differences in anatomy, embryological origin, and relation to aortic aneurysm. APPROACH AND RESULTS: The 2 other unique features of this study were (1) to study young (9±1 years) and old (26±1 years) male monkeys and (2) to study direct and continuous measurements of aortic pressure and thoracic and abdominal aortic diameters in conscious monkeys. As expected, aortic stiffness, ß, was increased P<0.05, 2- to 3-fold, in old versus young thoracic aorta and augmented further with superimposition of acute hypertension with phenylephrine. Surprisingly, stiffness was not greater in old thoracic aorta than in young abdominal aorta. These results can be explained, in part, by the collagen/elastin ratio, but more importantly, by disarray of collagen and elastin, which correlated best with vascular stiffness. However, vascular smooth muscle cell stiffness was not different in thoracic versus abdominal aorta in either young or old monkeys. CONCLUSIONS: Thus, aortic stiffness increases with aging as expected, but the most severe increases in aortic stiffness observed in the abdominal aorta is novel, where values in young monkeys equaled, or even exceeded, values of thoracic aortic stiffness in old monkeys. These results can be explained by alterations in collagen/elastin ratio, but even more importantly by collagen and elastin disarray.

GLP-1 (7-36) amide restores myocardial insulin sensitivity and prevents the progression of heart failure in senescent beagles.

BACKGROUND: We previously demonstrated that older beagles have impaired whole body and myocardial insulin responsiveness (MIR), and that glucagon-like peptide-1 (GLP-1 [7-36] amide) improves MIR in young beagles with dilated cardiomyopathy (DCM). Here, we sought to determine if aging alone predisposes to an accelerated course of DCM, and if GLP-1 [7-36] amide would restore MIR and impact the course of DCM in older beagles. METHODS: Eight young beagles (Young-Control) and sixteen old beagles underwent chronic left ventricle (LV) instrumentation. Seven old beagles were treated with GLP-1 (7-36) amide (2.5 pmol/kg/min) for 2 weeks prior to instrumentation and for 35 days thereafter (Old + GLP-1), while other 9 served as control (Old-Control). All dogs underwent baseline metabolic determinations and LV biopsy for mitochondria isolation prior to the development of DCM induced by rapid pacing (240 min-1). Hemodynamic measurements were performed routinely as heart failure progressed. RESULTS: At baseline, all old beagles had elevated non-esterifed fatty acids (NEFA), and impaired MIR. GLP-1 reduced plasma NEFA (Old-Control: 853 ± 34; Old + GLP-1: 531 ± 33 µmol/L, p < 0.02), improved MIR (Old-Control: 289 ± 54; Old + GLP-1: 512 ± 44 mg/min/100 mg, p < 0.05), and increased uncoupling protein-3 (UCP-3) expression in isolated mitochondria. Compared to the Young-Control, the Old-Controls experienced an accelerated course of DCM (7 days versus 29 days, p < 0.005) and excess mortality, while the Old + GLP-1 experienced increased latency to the onset of DCM (7 days versus 23 days, p < 0.005) and reduced mortality. CONCLUSION: Aging is associated with myocardial insulin resistance, which predispose to an accelerated course of DCM. GLP-1 treatment is associated with increased MIR and protection against an accelerated course of DCM in older beagles.

Smooth muscle myosin inhibition: a novel therapeutic approach for pulmonary hypertension.

OBJECTIVE: Pulmonary hypertension remains a major clinical problem despite current therapies. In this study, we examine for the first time a novel pharmacological target, smooth muscle myosin, and determine if the smooth muscle myosin inhibitor, CK-2019165 (CK-165) ameliorates pulmonary hypertension. MATERIALS AND METHODS: Six domestic female pigs were surgically instrumented to measure pulmonary blood flow and systemic and pulmonary vascular dynamics. Pulmonary hypertension was induced by hypoxia, or infusion of the thromboxane analog (U-46619, 0.1 µg/kg/min, i.v.). In rats, chronic pulmonary hypertension was induced by monocrotaline. RESULTS: CK-165 (4 mg/kg, i.v.) reduced pulmonary vascular resistance by 22±3 and 28±6% from baseline in hypoxia and thromboxane pig models, respectively (p<0.01 and 0.01), while mean arterial pressure also fell and heart rate rose slightly. When CK-165 was delivered via inhalation in the hypoxia model, pulmonary vascular resistance fell by 17±6% (p<0.05) while mean arterial pressure and heart rate were unchanged. In the monocrotaline model of chronic pulmonary hypertension, inhaled CK-165 resulted in a similar (18.0±3.8%) reduction in right ventricular systolic pressure as compared with sildenafil (20.3±4.5%). CONCLUSION: Inhibition of smooth muscle myosin may be a novel therapeutic target for treatment of pulmonary hypertension.

Cardiac myosin activation: a potential therapeutic approach for systolic heart failure.

Decreased cardiac contractility is a central feature of systolic heart failure. Existing drugs increase cardiac contractility indirectly through signaling cascades but are limited by their mechanism-related adverse effects. To avoid these limitations, we previously developed omecamtiv mecarbil, a small-molecule, direct activator of cardiac myosin. Here, we show that it binds to the myosin catalytic domain and operates by an allosteric mechanism to increase the transition rate of myosin into the strongly actin-bound force-generating state. Paradoxically, it inhibits adenosine 5'-triphosphate turnover in the absence of actin, which suggests that it stabilizes an actin-bound conformation of myosin. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure.

Preemptive conditioning of the swine heart by H11 kinase/Hsp22 provides cardiac protection through inducible nitric oxide synthase.

The second window of ischemic preconditioning (SWOP) provides maximal protection against ischemia through regulation of the inducible nitric oxide synthase (iNOS), yet its application is limited by the inconvenience of the preliminary ischemic stimulus required for prophylaxis. Overexpression of H11 kinase/Hsp22 (Hsp22) in a transgenic mouse model provides cardioprotection against ischemia that is equivalent to that conferred by SWOP. We hypothesized that short-term, prophylactic overexpression of Hsp22 would offer an alternative to SWOP in reducing ischemic damage through a nitric oxide (NO)-dependent mechanism. Adeno-mediated overexpression of Hsp22 was achieved in the area at risk of the left circumflex (Cx) coronary artery in chronically instrumented swine and compared with LacZ controls (n = 5/group). Hsp22-injected myocardium showed an average fourfold increase in Hsp22 protein expression compared with controls and a doubling in iNOS expression (both P < 0.05). Four days after ischemia-reperfusion, regional wall thickening was reduced by 58 ± 2% in the Hsp22 group vs. 82 ± 7% in the LacZ group, and Hsp22 reduced infarct size by 40% (both P < 0.05 vs. LacZ). Treatment with the NOS inhibitor N(G)-nitro-L-arginine (L-NNA) before ischemia suppressed the protection induced by Hsp22. In isolated cardiomyocytes, Hsp22 increased iNOS expression through the transcription factors NF-κB and STAT, the same effectors activated by SWOP, and reduced by 60% H(2)O(2)-mediated apoptosis, which was also abolished by NOS inhibitors. Therefore, short-term, prophylactic conditioning by Hsp22 provides NO-dependent cardioprotection that reproduces the signaling of SWOP, placing Hsp22 as a potential alternative for preemptive treatment of myocardial ischemia.

Primate models for cardiovascular drug research and development.

One of the primary impediments to successful drug R&D is the frequent failure of successfully translating positive results obtained in animal models to human disease. To a large degree, this discrepancy is secondary to the substantial biological differences between species. Non-human primate models have the advantage of significant physiological, metabolic, biochemical and genetic similarity to humans. Despite this advantage, there has been a relative paucity of non-human primate models used in the study of disease states that currently underlie the most common causes of morbidity and mortality, such as chronic myocardial ischemia leading to heart failure. This review describes a primate model of heart failure that closely mimics the cardiomyopathic process observed in humans. The primary advantage of this non-human primate model is that, unlike existing heart failure models, it allows for continuous study during progressive stages of heart failure, including myocardial ischemia, progressive left ventricular remodeling and end-stage congestive heart failure. In addition to this model of heart failure, other non-human primate models for cardiovascular drug R&D are also reviewed.

Molecular mechanisms mediating preconditioning following chronic ischemia differ from those in classical second window.

A major difference between experimental ischemic preconditioning (IPC), induced by brief ischemic episodes, and the clinical situation is that patients generally have repetitive episodes of ischemia. We used a swine model to examine differences in genes regulated by classical second-window IPC (SWOP) [two 10-min episodes of coronary artery occlusion (CAO) followed by 24 h reperfusion] compared with repetitive CAO/reperfusion (RCO), i.e., two 10-min CAO 12 h apart, and to repetitive coronary stenosis (RCS), six episodes of 90 min coronary stenosis 12 h apart (n = 5/group). All three models reduced infarct size by 60-85%, which was mediated by nitric oxide in SWOP but not in the other two models. We employed microarray analyses to discover additional molecular pathways intrinsic to models of repetitive ischemia and different from classical SWOP. There was an 85% homology in gene response between the RCO and RCS models, whereas SWOP was qualitatively different. Both RCO and RCS, but not SWOP, showed downregulation of genes encoding proteins involved in oxidative metabolism and upregulation of genes involved in protein synthesis, unfolded protein response, autophagy, heat shock response, protein secretion, and an activation of the NF-kappaB signaling pathway. Therefore, the regulated genes mediating IPC with repetitive ischemia differ radically from SWOP both quantitatively and qualitatively, showing that a repetitive pattern of ischemia, rather than the difference between no-flow vs. low-flow ischemia, dictates the genomic response of the heart. These findings illustrate new cardioprotective mechanisms developed by repetitive IPC, which are potentially more relevant to patients with chronic ischemic heart disease, who are subjected to repetitive episodes of ischemia.

Glucagon-like peptide-1 increases myocardial glucose uptake via p38alpha MAP kinase-mediated, nitric oxide-dependent mechanisms in conscious dogs with dilated cardiomyopathy.

BACKGROUND: We have shown that glucagon-like peptide-1 (GLP-1[7-36] amide) stimulates myocardial glucose uptake in dilated cardiomyopathy (DCM) independent of an insulinotropic effect. The cellular mechanisms of GLP-1-induced myocardial glucose uptake are unknown. METHODS AND RESULTS: Myocardial substrates and glucoregulatory hormones were measured in conscious, chronically instrumented dogs at control (n=6), DCM (n=9) and DCM after treatment with a 48-hour infusion of GLP-1 (7-36) amide (n=9) or vehicle (n=6). GLP-1 receptors and cellular pathways implicated in myocardial glucose uptake were measured in sarcolemmal membranes harvested from the 4 groups. GLP-1 stimulated myocardial glucose uptake (DCM: 20+/-7 nmol/min/g; DCM+GLP-1: 61+/-12 nmol/min/g; P=0.001) independent of increased plasma insulin levels. The GLP-1 receptors were upregulated in the sarcolemmal membranes (control: 98+/-2 density units; DCM: 256+/-58 density units; P=0.046) and were expressed in their activated (65 kDa) form in DCM. The GLP-1-induced increases in myocardial glucose uptake did not involve adenylyl cyclase or Akt activation but was associated with marked increases in p38alpha MAP kinase activity (DCM+vehicle: 97+/-22 pmol ATP/mg/min; DCM+GLP-1: 170+/-36 pmol ATP/mg/min; P=0.051), induction of nitric oxide synthase 2 (DCM+vehicle: 151+/-13 density units; DCM+GLP-1: 306+/-12 density units; P=0.001), and GLUT-1 translocation (DCM+vehicle: 21+/-3% membrane bound; DCM+GLP-1: 39+/-3% membrane bound; P=0.005). The effects of GLP-1 on myocardial glucose uptake were blocked by pretreatment with the p38alpha MAP kinase inhibitor or the nonspecific nitric oxide synthase inhibitor nitro-l-arginine. CONCLUSIONS: GLP-1 stimulates myocardial glucose uptake through a non-Akt-1-dependent mechanism by activating cellular pathways that have been identified in mediating chronic hibernation and the late phase of ischemic preconditioning.

Improvement of cardiac function by a cardiac Myosin activator in conscious dogs with systolic heart failure.

BACKGROUND: Therapy for chronic systolic heart failure (sHF) has improved over the past 2 decades, but the armamentarium of drugs is limited and consequently sHF remains a leading cause of death and disability. In this investigation, we examined the effects of a novel cardiac myosin activator, omecamtiv mecarbil (formerly CK-1827452) in 2 different models of heart failure. METHODS AND RESULTS: Two different models of sHF were used: (1) pacing-induced sHF after myocardial infarction (MI-sHF) and (2) pacing-induced sHF after 1 year of chronic pressure overload left ventricular hypertrophy (LVH-sHF). Omecamtiv mecarbil increased systolic function in sHF dogs, chronically instrumented to measure LV pressure, wall thickness, and cardiac output. Omecamtiv mecarbil, infused for 24 hours, induced a sustained increase without desensitization (P<0.05) in wall thickening (25+/-6.2%), stroke volume (44+/-6.5%) and cardiac output (22+/-2.8%), and decreased heart rate (15+/-3.0%). The major differences between the effect of omecamtiv mecarbil on cardiac function and the effect induced by a catecholamine, for example, dobutamine, is that omecamtiv mecarbil did not increase LV dP/dt but rather increased LV systolic ejection time by 26+/-2.9% in sHF. Another key difference is that myocardial O(2) consumption (MVO(2)), which increases with catecholamines, was not significantly affected by omecamtiv mecarbil. CONCLUSIONS: These results demonstrate that chronic infusion of the cardiac myosin activator, omecamtiv mecarbil, improves LV function in sHF without the limitations of progressive desensitization and increased MVO(2.) This unique profile may provide a new therapeutic approach for patients with sHF.

Effects of the prototype serotonin 5-HT(1B/1D) receptor agonist sumatriptan and the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37) on myocardial reactive hyperemic response in conscious dogs.

The triptans, serotonin 5-HT(1B/1D) receptor agonists exemplified by sumatriptan, are a mainstay migraine therapy but have class labeling contraindicating their use in patients with coronary artery disease. Triptans constrict human coronary artery in vitro, and there are case reports of myocardial infarction in patients using sumatriptan. However, preclinical studies with sumatriptan in normal dogs have failed to demonstrate effects on resting coronary flow. Calcitonin gene-related peptide (CGRP) receptor antagonism, exemplified by the prototype CGRP receptor antagonist peptide CGRP(8-37), is a new antimigraine mechanism which also has been reported to have no effect on coronary flow in normal, non-stressed animals. The goal of the present studies was to compare the effects of sumatriptan (10microg/kg/min i.v.) and CGRP(8-37) (30microg/kg/min i.v.) on systemic and coronary hemodynamics in conscious dogs under resting conditions and during myocardial reactive hyperemia following a brief 15s of coronary artery occlusion. Neither CGRP(8-37) nor sumatriptan affected resting coronary flow. However, whereas CGRP(8-37) had no effect on myocardial reactive hyperemic response, sumatriptan reduced peak reactive hyperemic coronary artery blood flow (baseline vs treatment: 75.4+/-12.7 vs 60.0+/-10.3ml/min, P<0.05), reactive hyperemic flow (16.7+/-5.2 vs 11.6+/-3.3ml, P<0.05) and the repayment of coronary blood flow debt following coronary artery occlusion (484+/-76 vs 369+/-57%, P<0.05), indicating an impairment in coronary blood flow reserve. The positive control nitric oxide synthase inhibitor L-NNA (30mg/kg/30min i.v.) likewise significantly attenuated myocardial reactive hyperemic response. These findings provide evidence for a differentiation between CGRP receptor antagonism and triptan effects on coronary vascular function.

Cytochrome c oxidase III as a mechanism for apoptosis in heart failure following myocardial infarction.

Cytochrome c oxidase (COX) is composed of 13 subunits, of which COX I, II, and III are encoded by a mitochondrial gene. COX I and II function as the main catalytic components, but the function of COX III is unclear. Because myocardial ischemia affects mitochondrial oxidative metabolism, we hypothesized that COX activity and expression would be affected during postischemic cardiomyopathy. This hypothesis was tested in a monkey model following myocardial infarction (MI) and subsequent pacing-induced heart failure (HF). In this model, COX I protein expression was decreased threefold after MI and fourfold after HF (P < 0.05 vs. sham), whereas COX II expression remained unchanged. COX III protein expression increased 5-fold after MI and further increased 10-fold after HF compared with sham (P < 0.05 vs. sham). The physiological impact of COX III regulation was examined in vitro. Overexpression of COX III in mitochondria of HL-1 cells resulted in an 80% decrease in COX I, 60% decrease in global COX activity, 60% decrease in cell viability, and threefold increase in apoptosis (P < 0.05). Oxidative stress induced by H2O2 significantly (P < 0.05) increased COX III expression. H2O2 decreased cell viability by 47 +/- 3% upon overexpression of COX III, but only by 12 +/- 5% in control conditions (P < 0.05). We conclude that ischemic stress in vivo and oxidative stress in vitro lead to upregulation of COX III, followed by downregulation of COX I expression, impaired COX oxidative activity, and increased apoptosis. Therefore, upregulation of COX III may contribute to the increased susceptibility to apoptosis following MI and subsequent HF.

Apoptosis predominates in nonmyocytes in heart failure.

The goal of this investigation was to determine the distribution of myocardial apoptosis in myocytes and nonmyocytes in primates and patients with heart failure (HF). Almost all clinical cardiologists and cardiovascular investigators believe that myocyte apoptosis is considered to be a cardinal sign of HF and a major factor in its pathogenesis. However, with the knowledge that 75% of the number of cells in the heart are nonmyocytes, it is important to determine whether the apoptosis in HF is occurring in myocytes or in nonmyocytes. We studied both a nonhuman primate model of chronic HF, induced by rapid pacing 2-6 mo after myocardial infarction (MI), and biopsies from patients with ischemic cardiomyopathy. Dual labeling with a cardiac muscle marker was used to discriminate apoptosis in myocytes versus nonmyocytes. Left ventricular ejection fraction decreased following MI (from 78% to 60%) and further with HF (35%, P < 0.05). As expected, total apoptosis was increased in the myocardium following recovery from MI (0.62 cells/mm(2)) and increased further with the development of HF (1.91 cells/mm(2)). Surprisingly, the majority of apoptotic cells in MI and MI + HF, and in both the adjacent and remote areas, were nonmyocytes. This was also observed in myocardial biopsies from patients with ischemic cardiomyopathy. We found that macrophages contributed the largest fraction of apoptotic nonmyocytes (41% vs. 18% neutrophils, 16% fibroblast, and 25% endothelial and other cells). Although HF in the failing human and monkey heart is characterized by significant apoptosis, in contrast to current concepts, the apoptosis in nonmyocytes was eight- to ninefold greater than in myocytes.

Repetitive ischemia by coronary stenosis induces a novel window of ischemic preconditioning.

BACKGROUND: The hypothesis of the present study was that molecular mechanisms differ markedly when mediating ischemic preconditioning induced by repetitive episodes of ischemia versus classic first- or second-window preconditioning. METHODS AND RESULTS: To test this, chronically instrumented conscious pigs were subjected to either repetitive coronary stenosis (RCS) or a traditional protocol of second-window ischemic preconditioning (SWIPC). Lethal ischemia, induced by 60 minutes of coronary artery occlusion followed by reperfusion, resulted in an infarct size/area at risk of 6+/-3% after RCS and 16+/-3% after SWIPC (both groups P<0.05, less than shams 42+/-4%). Two molecular signatures of SWIPC, the increased expression of the inducible isoform of nitric oxide synthase and the translocation of protein kinase Cepsilon to the plasma membrane, were observed with SWIPC but not with RCS. Confirming this, pretreatment with a nitric oxide synthase inhibitor prevented the protection conferred by SWIPC but not by RCS. Microarray analysis revealed a qualitatively different genomic profile of cardioprotection between ischemic preconditioning induced by RCS and that induced by SWIPC. The number of genes significantly regulated was greater in RCS (5739) than in SWIPC (2394) animals. Of the 5739 genes regulated in RCS, only 31% were also regulated in SWIPC. Broad categories of genes induced by RCS but not SWIPC included those involved in autophagy, endoplasmic reticulum stress, and mitochondrial oxidative metabolism. The upregulation of these pathways was confirmed by Western blotting. CONCLUSIONS: RCS induces cardioprotection against lethal myocardial ischemia that is at least as powerful as traditional ischemic preconditioning but is mediated through radically different mechanisms.

Cardiac dysfunction in aging conscious rats: altered cardiac cytoskeletal proteins as a potential mechanism.

The objective of this study was to test the hypothesis that the mechanism mediating left ventricular (LV) dysfunction in the aging rat heart involves, in part, changes in cardiac cytoskeletal components. Our results show that there were no significant differences in heart rate, LV pressure, or LV diameter between conscious, instrumented young [5.9 +/- 0.3 mo (n = 9)] and old rats [30.6 +/- 0.1 mo (n = 10)]. However, the first derivative of LV pressure (LV dP/dt) was reduced (8,309 +/- 790 vs. 11,106 +/- 555 mmHg/s, P < 0.05) and isovolumic relaxation time (tau) was increased (8.7 +/- 0.7 vs. 6.3 +/- 0.6 ms, P < 0.05) in old vs. young rats, respectively. The differences in baseline LV function in young and old rats, which were modest, were accentuated after beta-adrenergic receptor stimulation with dobutamine (20 mug/kg), which increased LV dP/dt by 170 +/- 9% in young rats, significantly more (P < 0.05) than observed in old rats (115 +/- 5%). Volume loading in anesthetized rats demonstrated significantly impaired LV compliance in old rats, as measured by the LV end-diastolic pressure and dimension relationship. In old rat hearts, there was a significant (P < 0.05) increase in the percentage of LV collagen (2.4 +/- 0.2 vs. 1.3 +/- 0.2%), alpha-tubulin (92%), and beta-tubulin (2.3-fold), whereas intact desmin decreased by 51%. Thus the cardiomyopathy of aging in old, conscious rats may be due not only to increases in collagen but also to alterations in cytoskeletal proteins.

Aging is associated with myocardial insulin resistance and mitochondrial dysfunction.

Aging is associated with insulin resistance, often attributable to obesity and inactivity. Recent evidence suggests that skeletal muscle insulin resistance in aging is associated with mitochondrial alterations. Whether this is true of the senescent myocardium is unknown. Twelve young (Y, 4 years old) and 12 old (O, 11 years old) dogs, matched for body mass, were instrumented with left-ventricular pressure gauges, aortic and coronary sinus catheters, and flow probes on left circumflex artery. Before surgery, all dogs participated in a 6-wk exercise program. Dogs underwent measurements of hemodynamics and plasma substrates before and during a 2-h hyperinsulinemic-euglycemic clamp to measure whole body and myocardial glucose and nonesterified fatty acid uptake. Following the protocol, myocardial and skeletal samples were obtained to measure components of the insulin-signaling cascade and mitochondrial structure. There was no difference in plasma glucose (Y, 90 +/- 4 mg/dl; O, 87 +/- 4 mg/dl), but old dogs had higher (P < 0.02) nonesterified fatty acids (Y, 384 +/- 48 micromol/l; O, 952 +/- 97 micromol/l) and plasma insulin (Y, 39 +/- 11 pmol/l; O, 108 +/- 18 pmol/l). Old dogs had impaired total body glucose disposition (Y, 11.5 +/- 1 mg x kg(-1) x min(-1); O, 8.0 +/- 0.5 mg x kg(-1) x min(-1); P < 0.05) and insulin-stimulated myocardial glucose uptake (Y, 3.5 +/- 0.3 mg x min(-1) x g(-1); O, 1.8 +/- 0.3 mg x min(-1) x g(-1); P < 0.05). The impaired insulin action was associated with altered insulin signaling and glucose transporter (GLUT4) translocation. There were myocardial mitochondrial structural changes observed in association with decreased expression of uncoupling protein-3. Aging is associated with both whole body and myocardial insulin resistance, independent of obesity and inactivity, but involving altered mitochondrial structure and impaired cellular insulin action.

Increased apoptosis and myocyte enlargement with decreased cardiac mass; distinctive features of the aging male, but not female, monkey heart.

We studied gender-specific changes in aging cardiomyopathy in a primate model, Macaca fascicularis, free of the major human diseases, complicating the interpretation of data specific to aging in humans. Left ventricular (LV) weight/body weight decreased, p<0.05, in old males but did not change in old females. However, despite the decrease in LV weight, mean myocyte cross-sectional area in the old males increased by 51%. This increase in myocyte size was not uniform in old males, i.e., it was manifest in only 20-30% of all the myocytes from old males. In old males there was a 4-fold increase in frequency of myocyte apoptosis without any increase in proliferation-capable myocytes assessed by Ki-67 expression. Apoptosis was unchanged in old female monkey hearts, whereas the frequency of myocytes expressing Ki-67 declined 90%. These results, opposite to findings from rodent studies, indicate distinct differences in which male and female monkeys maintain functional heart mass during aging. The old male hearts demonstrated increased apoptosis, which more than offset the myocyte hypertrophy. Interestingly, the hypertrophy was not uniform and there was no significant increase in myocyte proliferation.

Mechanism of gender-specific differences in aortic stiffness with aging in nonhuman primates.

BACKGROUND: Our hypothesis was that the changes in vascular properties responsible for aortic stiffness with aging would be greater in old male monkeys than old female monkeys. METHODS AND RESULTS: We analyzed the effects of gender differences in aging on in vivo measurements of aortic pressure and diameter and on extracellular matrix of the thoracic aorta in young adult (age, 6.6+/-0.5 years) versus old adult (age, 21.2+/-0.2 years) monkeys (Macaca fascicularis). Aortic stiffness, as represented by the pressure strain elastic modulus (Ep), increased more in old male monkeys (5.08+/-0.81; P<0.01) than in old females (3.06+/-0.52). In both genders, collagen density was maintained, collagen-bound glycation end products increased, and collagen type 1 decreased. However, elastin density decreased significantly (from 22+/-1.5% to 15+/-1.2%) with aging (P<0.05) only in males. Furthermore, only old males were characterized by a decrease (P<0.05) in collagen type 3 (an isoform that promotes elasticity) and an increase in collagen type 8 (an isoform that promotes the neointimal migration of vascular smooth muscle cells). In contrast to the data in monkeys, collagen types 1 and 3 both increased significantly in aging rats. CONCLUSIONS: There are major species differences in the effects of aging on aortic collagen types 1 and 3. Furthermore, because alterations in collagen density, collagen content, hydroxyproline, and collagen advanced glycation end products were similar in both old male and female monkeys, these factors cannot be responsible for the greater increase in stiffness in old males. However, changes in collagen isoforms and the decrease in elastin observed only in old males likely account for the greater increase in aortic stiffness.

Sex-specific regulation of gene expression in the aging monkey aorta.

Although increased vascular stiffness is more prominent in aging males than females, and males are more prone to vascular disease with aging, no study has investigated the genes potentially responsible for sex differences in vascular aging. We tested the hypothesis that the transcriptional adaptation to aging differs in males and females using a monkey model, which is not only physiologically and phylogenetically closer to humans than the more commonly studied rodent models but also is not afflicted with the most common forms of vascular disease that accompany the aging process in humans, e.g., atherosclerosis, hypertension, and diabetes. The transcriptional profile of the aorta was compared by high-density microarrays between young and old males or females (n = 6/group). About 600 genes were expressed differentially when comparing old versus young animals. Surprisingly, <5% of these genes were shared between males and females. Radical differences between sexes were especially apparent for genes regulating the extracellular matrix, which relates to stiffness. Aging males were also more prone than females to genes switching smooth muscle cells from the "contractile" to "secretory" phenotype. Other sex differences involved genes participating in DNA repair, stress response, and cell signaling. Therefore, major differences of gene regulation exist between males and females in vascular aging, which may underlie the physiological differences characterizing aging arteries in males and females. Furthermore, the analyses in young monkeys demonstrated differences in genes regulating vascular structure, implying that the sex differences in vascular stiffness that develop with aging are programmed at an early age.

Altered autonomic control in conscious transgenic rabbits with overexpressed cardiac Gsalpha.

Both enhanced sympathetic drive and altered autonomic control are involved in the pathogenesis of heart failure. The goal of the present study was to determine the extent to which chronically enhanced sympathetic drive, in the absence of heart failure, alters reflex autonomic control in conscious, transgenic (TG) rabbits with overexpressed cardiac Gsalpha. Nine TG rabbits and seven wild-type (WT) littermates were instrumented with a left ventricular (LV) pressure micromanometer and arterial catheters and studied in the conscious state. Compared with WT rabbits, LV function was enhanced in TG rabbits, as reflected by increased levels of LV dP/dt (5,600 +/- 413 vs. 3,933 +/- 161 mmHg/s). Baseline heart rate was also higher (P < 0.05) in conscious TG (247 +/- 10 beats/min) than in WT (207 +/- 10 beats/min) rabbits and was higher in TG after muscarinic blockade (281 +/- 9 vs. 259 +/- 8 beats/min) or combined beta-adrenergic receptor and muscarinic blockade (251 +/- 6 vs. 225 +/- 9 beats/min). Bradycardia was blunted (P < 0.05), whether induced by intravenous phenylephrine (arterial baroreflex), by cigarette smoke inhalation (nasopharyngeal reflex), or by veratrine administration (Bezold-Jarisch reflex). With veratrine administration, the bradycardia was enhanced in TG for any given decrease in arterial pressure. Thus the chronically enhanced sympathetic drive in TG rabbits with overexpressed cardiac Gsalpha resulted in enhanced LV function and heart rate and impaired reflex autonomic control. The impaired reflex control was generalized, not only affecting the high-pressure arterial baroreflex but also the low-pressure Bezold-Jarisch reflex and the nasopharyngeal reflex.

Obligatory role of cardiac nerves and alpha1-adrenergic receptors for the second window of ischemic preconditioning in conscious pigs.

We tested the hypothesis that cardiac nerves may mediate ischemic preconditioning. Pigs were chronically instrumented to measure aortic, left atrial and left ventricular pressures, and regional myocardial function (wall thickening). Hemodynamic variables, area at risk, and tissue blood flows (radioactive microspheres) were similar among groups. Myocardial infarct size following 60 minutes coronary artery occlusion and 4 days reperfusion, expressed as a fraction of the area at risk, was 42+/-4.0%, in innervated pigs and similar in pigs with regional cardiac denervation (CD, 41+/-2.5%). Infarct size in innervated pigs during the first window of preconditioning (first window) was markedly reduced (6+/-1.8%, P<0.01), as it was in the second window of preconditioning (second window) (16+/-3.3%, P<0.01). Although infarct size was still reduced in pigs with CD and first window preconditioning (9+/-1.8%, P<0.01), the protective effects of second window were abrogated in pigs with CD resulting in an infarct size of 38+/-5.6%. In another group of innervated pigs during pharmacological alpha(1)-adrenergic receptor (AR) blockade, infarct size was also not reduced during the second window (48+/-3.2%). Additionally, Western blot analysis of inducible nitric oxide synthase and cyclooxygenase-2 proteins demonstrated significant (P<0.05) upregulation following the second window in innervated pigs, but not in pigs with CD or alpha(1)-AR blockade. Thus, the mechanism of protection during the second window, but not the first window, appears to be dependent on cardiac nerves and alpha(1)-AR stimulation.