Melissa Krawczyk

Erythropoietin reduces myocardial infarction and left ventricular functional decline after coronary artery ligation in rats

Erythropoietin (EPO), well known for its role in stimulation of erythropoiesis, has recently been shown to have a dramatic neuroprotective effect in animal models of cerebral ischemia, mechanical trauma of the nervous system, and excitotoxins, mainly by reducing apoptosis. We studied the effect of single systemic administration of recombinant human EPO (rhEPO) on left ventricular (LV) size and function in rats during 8 weeks after the induction of a myocardial infarction (MI) by permanent ligation of the left descending coronary artery. We found that an i.p. injection of 3,000 units/kg of rhEPO immediately after the coronary artery ligation resulted, 24 h later, in a 50% reduction of apoptosis in the myocardial area at risk. Eight weeks after the induction of MI, rats treated with rhEPO had an infarct size 15–25% of the size of that in untreated animals. The reduction in myocardial damage was accompanied by reductions in LV size and functional decline as measured by repeated echocardiography. Thus, a single dose of rhEPO administered around the time of acute, sustained coronary insufficiency merits consideration with respect to its therapeutic potential to limit the extent of resultant MI and contractile dysfunction.

SRT1720 improves survival and healthspan of obese mice

Sirt1 is an NAD+-dependent deacetylase that extends lifespan in lower organisms and improves metabolism and delays the onset of age-related diseases in mammals. Here we show that SRT1720, a synthetic compound that was identified for its ability to activate Sirt1 in vitro, extends both mean and maximum lifespan of adult mice fed a high-fat diet. This lifespan extension is accompanied by health benefits including reduced liver steatosis, increased insulin sensitivity, enhanced locomotor activity and normalization of gene expression profiles and markers of inflammation and apoptosis, all in the absence of any observable toxicity. Using a conditional SIRT1 knockout mouse and specific gene knockdowns we show SRT1720 affects mitochondrial respiration in a Sirt1- and PGC-1α-dependent manner. These findings indicate that SRT1720 has long-term benefits and demonstrate for the first time the feasibility of designing novel molecules that are safe and effective in promoting longevity and preventing multiple age-related diseases in mammals.

Angiotensin II activates matrix metalloproteinase type II and mimics age-associated carotid arterial remodeling in young rats.

Increased angiotensin II (Ang II), matrix metalloproteinase type II (MMP2), and sympathetic activity accompany age-associated arterial remodeling. To analyze this relationship, we infused a low subpressor dose of Ang II into young (8 months old) rats. This increased carotid arterial MMP2 transcription, translation, and activation, as well as transforming growth factor-beta1 activity and collagen deposition. A higher Ang II concentration, which increased arterial pressure to that of old (30 months old) untreated rats, produced carotid media thickening and intima infiltration by vascular smooth muscle cells (VSMCs). Ex vivo, Ang II increased MMP2 activity in carotid rings from young rats to that of untreated old rats. Ang II also increased the ability of early passage VSMCs from young rats to invade a synthetic basement membrane, similar to that of untreated VSMCs from old rats. The MMP inhibitor GM6001 and the AT1 receptor antagonist Losartan inhibited these effects. The alpha-adrenoreceptor agonist phenylephrine increased arterial Ang II protein, causing MMP2 activation and intima and media thickening. Exposure of young VSMCs to phenylephrine in vitro increased Ang II protein and MMP2 activity to the levels of old VSMCs; Losartan abolished these effects. Thus, Ang II-induced effects on MMP2, transforming growth factor-beta1, collagen, and VSMCs are central to the arterial remodeling that accompanies advancing age.

Beneficial Effects of Chronic Pharmacological Manipulation of β-Adrenoreceptor Subtype Signaling in Rodent Dilated Ischemic Cardiomyopathy

Background—Studies in isolated cardiac myocytes have demonstrated that signaling via specific &bgr;1-adrenergic receptor subtypes (&bgr;1ARs) promotes but that signaling via &bgr;2ARs protects from cell death. We hypothesized that prolonged &bgr;2AR stimulation or &bgr;1AR blockade would each protect myocytes from death and thereby ameliorate cardiac remodeling in chronic heart failure. Methods and Results—A large myocardial infarction (MI) induced in rats by coronary artery ligation resulted in a dilated cardiomyopathy (DCM) characterized by infarct expansion and a progressive increase in left ventricular (LV) end-diastolic volume, accompanied by a reduction in ejection fraction (EF), as assessed by repeated echocardiography. Pressure-volume analysis at 8 weeks after ligation showed that diastolic stiffness (Eed) and arterial elastance (Ea) were increased, end-systolic elastance (Ees) was decreased, and arterioventricular (AV) coupling (Ea/Ees) had deteriorated. Apoptosis was present in both peri-infarct and remote myocardium. Chronic (6-week) administration of the &bgr;2AR agonists fenoterol or zinterol, starting at 2 weeks after MI, reduced the extent of LV dilation, infarct expansion, and EF decline. The &bgr;1AR blocker metoprolol did not affect the former and preserved EF to a lesser extent than did the &bgr;2AR agonists. At 8 weeks after ligation, apoptosis was reduced by all drugs but to a greater extent by &bgr;2AR agonists than by the &bgr;1AR blocker. Both &bgr;2AR agonists and the &bgr;1AR blocker improved AV coupling, the former mainly by reducing Ea and the latter mainly by increasing Ees. Only the &bgr;2AR agonists reduced the Eed and the MI size by reducing infarct expansion. Conclusions—These results provide proof of concept for the efficacy of chronic &bgr;2AR stimulation in this DCM model.

Cardioprotective and Survival Benefits of Long-Term Combined Therapy with β2 Adrenoreceptor (AR) Agonist and β1 AR Blocker in Dilated Cardiomyopathy Postmyocardial Infarction

We have reported therapeutic effectiveness of pharmacological stimulation of β2 adrenoreceptors (ARs) to attenuate the cardiac remodeling and myocardial infarction (MI) expansion in a rat model of dilated cardiomyopathy (DCM) post-MI. Furthermore, the combination of β2 AR stimulation with β1 AR blockade exceeded the therapeutic effectiveness of β1 AR blockade. However, these studies were relatively short (6 weeks). In this study, in the same experimental model, we compared different effects, including survival benefit, of combined therapy with the β1 AR blocker, metoprolol, plus the β2 AR agonist, fenoterol (β1–β2+), and either therapy alone (β1– or β2+) during the 1-year study. Therapy was started 2 weeks after permanent ligation of the left coronary artery. Cardiac remodeling, MI expansion, and left ventricular function were assessed by serial echocardiography and compared with untreated animals (nT). Sixty-seven percent mortality in nT was reduced to 33% in the β1–β2+ (p < 0.01). Progressive cardiac remodeling observed in nT and β1– was significantly attenuated in β1–β2+ during the first 6 months of treatment. In β1–β2+, MI expansion was completely prevented, and functional decline was significantly attenuated during the entire year. Myocardial apoptosis was significantly reduced in both β1–β2+ and β1–. A reduction of cardiac β1 AR density and decreases in chronotropic and contractile responses to β2 AR-specific stimulation in the absence of a reduction of β2 AR density in nT were precluded in rats receiving combined therapy. The results demonstrate the cardioprotective and survival benefit of long-term combination therapy of β2 AR agonists and β1 AR blockers in a model of DCM.

The pro-angiogenic cytokine pleiotrophin potentiates cardiomyocyte apoptosis through inhibition of endogenous AKT/PKB activity.

Pleiotrophin is a development-regulated cytokine and growth factor that can promote angiogenesis, cell proliferation, or differentiation, and it has been reported to have neovasculogenic effects in damaged heart. Developmentally, it is prominently expressed in fetal and neonatal hearts, but it is minimally expressed in normal adult heart. Conversely, we show in a rat model of myocardial infarction and in human dilated cardiomyopathy that pleiotrophin is markedly up-regulated. To elucidate the effects of pleiotrophin on cardiac contractile cells, we employed primary cultures of rat neonatal and adult cardiomyocytes. We show that pleiotrophin is released from cardiomyocytes in vitro in response to hypoxia and that the addition of recombinant pleiotrophin promotes caspase-mediated genomic DNA fragmentation in a dose- and time-dependent manner. Functionally, it potentiates the apoptotic response of neonatal cardiomyocytes to hypoxic stress and to ultraviolet irradiation and of adult cardiomyocytes to hypoxia-reoxygenation. Moreover, UV-induced apoptosis in neonatal cardiomyocytes can be partially inhibited by small interfering RNA-mediated knockdown of endogenous pleiotrophin. Mechanistically, pleiotrophin antagonizes IGF-1 associated Ser-473 phosphorylation of AKT/PKB, and it concomitantly decreases both BAD and GSK3β phosphorylation. Adenoviral expression of constitutively active AKT and lithium chloride-mediated inhibition of GSK3β reduce the potentiated programmed cell death elicited by pleiotrophin. These latter data indicate that pleiotrophin potentiates cardiomyocyte cell death, at least partially, through inhibition of AKT signaling. In conclusion, we have uncovered a novel function for pleiotrophin on heart cells following injury. It fosters cardiomyocyte programmed cell death in response to pro-apoptotic stress, which may be critical to myocardial injury repair.

Cardiac remodeling in the mouse model of Marfan syndrome develops into two distinctive phenotypes

Marfan syndrome (MFS) is a systemic disorder of connective tissue caused by mutations in fibrillin-1. Cardiac dysfunction in MFS has not been characterized halting the development of therapies of cardiac complication in MFS. We aimed to study the age-dependent cardiac remodeling in the mouse model of MFS FbnC1039G+/- mouse [Marfan heterozygous (HT) mouse] and its association with valvular regurgitation. Marfan HT mice of 2-4 mo demonstrated a mild hypertrophic cardiac remodeling with predominant decline of diastolic function and increased transforming growth factor-β canonical (p-SMAD2/3) and noncanonical (p-ERK1/2 and p-p38 MAPK) signaling and upregulation of hypertrophic markers natriuretic peptides atrium natriuretic peptide and brain natriuretic peptide. Among older HT mice (6-14 mo), cardiac remodeling was associated with two distinct phenotypes, manifesting either dilated or constricted left ventricular chamber. Dilatation of left ventricular chamber was accompanied by biochemical evidence of greater mechanical stress, including elevated ERK1/2 and p38 MAPK phosphorylation and higher brain natriuretic peptide expression. The aortic valve regurgitation was registered in 20% of the constricted group and 60% of the dilated group, whereas mitral insufficiency was observed in 40% of the constricted group and 100% of the dilated group. Cardiac dysfunction was not associated with the increase of interstitial fibrosis and nonmyocyte proliferation. In the mouse model fibrillin-1, haploinsufficiency results in the early onset of nonfibrotic hypertrophic cardiac remodeling and dysfunction, independently from valvular abnormalities. MFS heart is vulnerable to stress-induced cardiac dilatation in the face of valvular regurgitation, and stress-activated MAPK signals represent a potential target for cardiac management in MFS.

Doxycycline ameliorates the susceptibility to aortic lesions in a mouse model for the vascular type of Ehlers-Danlos syndrome

The vascular form of Ehlers-Danlos syndrome (vEDS), a rare disease with grave complications resulting from rupture of major arteries, is caused by mutations of collagen type III [α1 chain of collagen type III (COL3A1)]. The only, recently proven, preventive strategy consists of the reduction of arterial wall stress by β-adrenergic blockers. The heterozygous (HT) Col3a1 knockout mouse has reduced expression of collagen III and recapitulates features of a mild presentation of the disease. The objective of this study was to determine whether changing the balance between synthesis and degradation of collagen by chronic treatment with doxycycline, a nonspecific matrix metalloproteinase (MMP) inhibitor, could prevent the development of vascular pathology in HT mice. After 3 months of treatment with doxycycline or placebo, 9-month-old HT or wild-type (WT) mice were subjected to surgical stressing of the aorta. A 3-fold increase in stress-induced aortic lesions found in untreated HT mice 1 week after intervention (cumulative score 4.5 ± 0.87 versus 1.3 ± 0.34 in WT, p < 0.001) was fully prevented in the doxycycline-treated group (1.1 ± 0.56, p < 0.001). Untreated HT mice showed increased MMP-9 activity in the carotid artery and decreased collagen content in the aorta; however, in doxycycline-treated animals there was normalization to the levels observed in WT mice. Doxycycline treatment inhibits the activity of tissue MMP and attenuates the decrease in the collagen content in aortas of mice haploinsufficient for collagen III, as well as prevents the development of stress-induced vessel pathology. The results suggest that doxycycline merits clinical testing as a treatment for vEDS.

Cardiac Remodeling in the Mouse Model of Marfan Syndrome Develops Independently from Aortic and Valvular Abnormalities

Marfan syndrome (MFS) is a multisystem disorder of connective tissue caused by mutations in fibrillin-1. Heart involvement in the Marfan syndrome patients includes aortic root dilatation, valvular insufficiency, and myocardial dysfunction; it remains unclear, however, whether alterations in myocardium are triggered by valvular and aortic pathology or they develop independently. We evaluated the age-dependent cardiac remodeling and left ventricular dysfunction in the mouse model of MFS known as Fbn1039G+/− mouse (Marfan HT mouse) using echocardiography, pressure-volume loop analyses and a number of histological and biochemical techniques.Marfan HT mice of 2-4 month demonstrated a hypertrophic cardiac remodeling accompanied by predominant decline of diastolic function and increased TGF-β canonical (p SMAD2/3) and non-canonical (pERK ½ and pMAPK38) signaling. Hypertrophic myocardium among older HT mice (6-14 months) was associated with two distinctly different phenotypes manifesting either dilated or constricted LV chamber. Dilatation of LV chamber was accompanied by biochemical evidence of greater mechanical stress, including elevated ERK1/2 phosphorylation and brain natriuretic peptide expression in comparison with constricted heart. Diastolic dysfunction in the older HT mice was combined with significant systolic impairment. The aortic valve regurgitation was registered in 20% of constricted group and 60% of dilated, while mitral insufficiency was observed in 40% of constricted group and 100% of dilated. In Marfan HT mice, extracellular matrix abnormalities were not associated with the increase of interstitial fibrosis and non-myocyte proliferation. In the mouse model of fibrillin-1 haploinsufficiency the early onset of hypertrophic cardiac remodeling and dysfunction is not consequent to functional valvular abnormalities, but it is likely to result from deficient mechanosensing and transmission of mechanical forces.

Complimentary effects of chronic pharmacologic manipulation of β-adrenergic receptor (βAR) subtype signaling in rodent dilated ischemic cardiomyopathy

Background: While treatment of dilated cardiomyopathy with β1AR blockers has proven beneficial, the potential added, well-documented benefits of β2AR receptor stimulation, e.g. protection from apoptosis and reduction of arterial elastance with respect to chronic heart failure, have not been addressed. Method: A myocardial infarction (MI) was effected by left descending coronary artery ligation in 88 rats. Five rats were subjected to sham operation (SH). One week later the ligated animals were randomized to treatment groups, insuring equal initial MI size (27% of LV endocardial circumference at end-diastole in echocardiographic short-axis view at mid-papillary muscle level) among the groups: untreated (UT) MI; β1AR blocker, Metoprolol; and β2AR agonist, Fenoterol. Treatment dissolved in drinking water starting from 2 weeks post MI. Following 6 weeks of treatment LV end-systolic elastance, end-diastolic stiffness, and arterial elastance (Ees, Eed, Ea), arterio-ventricular (AV) coupling (Ea/Ees), Ejection Fraction (EF), end-diastolic volume (EDV) and endsystolic volume (ESV) were assessed by LV pressure-volume loops analyses or echocardiography. Hearts were then harvested for histological evaluation of MI size and TUNEL staining for apoptosis (APOP) in the MI border zone and in remote area. Results: The large MI in UT produced a dilated ischemic cardiomyopathy with increases (compared to SH) in EDV and ESV and reduction in EF. Ees was reduced and Ea increased resulting in AV mismatch (increase in Ea/Ees). APOP also was substantially increased (Table). βAR subtypes treatment both improved EF and AV coupling, but by different mechanism: β1AR blocker increased Ees without effect on Ea; β2AR agonist reduced Ea without effect on Ees. Only β2AR agonist reduced EDV and ESV and Eed. β2AR stimulation reduced apoptosis to greater extent than β1AR blocker and only β2AR stimulation reduced theMI size.Conclusion. β1AR blockade and β2AR stimulation had differing hemodynamic effects that both improved AV coupling and LV pump function. While both also reduced apoptosis, only β2AR stimulation reduced the heart size and extent of the MI. The complimentary beneficial effects of the β1AR blockers and β2AR agonists on the LV and arterial system provides rationale for their combined usage, at least with respect to amelioration of dilated ischemic cardiomyopathy.