[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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%).
Inhibition of smooth muscle myosin may be a novel therapeutic target for treatment of pulmonary hypertension.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Feb 2011 · AJP Heart and Circulatory Physiology
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
No preview · Article · May 2010 · Circulation Heart Failure
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · May 2009 · AJP Heart and Circulatory Physiology
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
RCS induces cardioprotection against lethal myocardial ischemia that is at least as powerful as traditional ischemic preconditioning but is mediated through radically different mechanisms.
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Jun 2008 · AJP Heart and Circulatory Physiology
[Show abstract][Hide abstract] ABSTRACT: 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.
Full-text · Article · Oct 2007 · Journal of Molecular and Cellular Cardiology
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Mar 2007 · AJP Heart and Circulatory Physiology
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Dec 2006 · Circulation Research
[Show abstract][Hide abstract] ABSTRACT: We overexpressed cardiac Gsalpha in rabbits using the beta-myosin heavy chain promoter. Gsalpha protein levels in the heart were increased 3-fold by Western blotting in both juvenile (3-4 months), adult (8-10 months), and older (11-16 months) rabbits, compared with wild type (WT) littermates. In transgenic (TG) rabbits, baseline levels of heart rate were elevated, P<0.05 (268+/-17 vs. 209+/-15 beats/min), as well as left ventricular (LV) contractility (LV dP/dt 5475+/-482 vs. 3740+/-246 mm Hg/s). These values and LV ejection fraction remained significantly elevated in older TG rabbits (11-16 months). However, maximal levels of LV dP/dt and heart rate with a high dose of isoproterenol (0.4 microg/kg/min) were similar in adult TG and WT rabbits. In isolated myocytes from the LV of adult rabbits, baseline percent contraction was increased, P<0.05, in TG (11.2+/-0.5%) compared to WT (9.3+/-0.5%), while maximal responses to isoproterenol (100 nM) were similar in adult TG (16.2+/-0.5%) and WT myocytes (15.6+/-0.4%). Although TG mice with overexpressed cardiac Gsalpha develop cardiomyopathy at 8-12 months of age, even at 16 months of age, there was no evidence of cardiomyopathy either in terms of LV function or histology in TG rabbits. In addition, Gialpha was elevated in the LV of adult (8-10 months old) TG rabbits compared to WT, but not in juvenile (3-5 months old) TG rabbits. Although both TG mice and rabbits with overexpressed cardiac Gsalpha exhibited enhanced heart rate and contractility, the TG rabbit does not develop cardiomyopathy, potentially due to a compensatory increase in Gialpha.
No preview · Article · Jul 2006 · Journal of Molecular and Cellular Cardiology
[Show abstract][Hide abstract] ABSTRACT: We examined the effects of gender and aging on cardiac and peripheral hemodynamic responses to beta-adrenergic receptor (beta-AR) stimulation in young (male = 5.9 +/- 0.4 yr old and female = 6.5 +/- 0.7 yr old) and old (male = 19.8 +/- 0.7 yr old and female = 21.2 +/- 0.2 yr old) conscious monkeys (Macaca fascicularis), chronically instrumented for measurements of left ventricular (LV) and arterial pressures as well as cardiac output. Baseline LV pressure, the first derivative of LV pressure (LV dP/dt), cardiac index, mean arterial pressure, total peripheral resistance (TPR), and heart rate in conscious monkeys were not different among the four groups. Increases in LV dP/dt in response to 0.1 microg/kg isoproterenol (Iso) were diminished (P < 0.05) in old males (+99 +/- 11%) compared with young males (+194 +/- 18%). In addition, the inotropic responses to norepinephrine (NE) and forskolin (FSK) were significantly depressed (P < 0.05) in old males. Iso-induced reductions of TPR were less (P < 0.05) in old males (-28 +/- 2%) than in young males (-49 +/- 2%). The changes of TPR in response to NE and FSK were also significantly attenuated (P < 0.05) in old males. However, the LV dP/dt responses to BAY y 5959 (15 microg. kg-1. min-1), a Ca2+ channel promotor independent of beta-AR signaling, were not significantly different between old and young males. In contrast to results in male monkeys, LV dP/dt and TPR responses to Iso, NE, and FSK in old females were similar to those observed in young females. Thus both cardiac contractile and peripheral vascular dynamic responses to beta-AR stimulation are preserved in old female but not old male monkeys. This may explain, in part, the reduced cardiovascular risk in the older female population.
Full-text · Article · Aug 2003 · AJP Heart and Circulatory Physiology