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1.  Angiotensin-converting enzyme inhibition and food restriction restore delayed preconditioning in diabetic mice 
Classical and delayed preconditioning are powerful endogenous protection mechanisms against ischemia-reperfusion damage. However, it is still uncertain whether delayed preconditioning can effectively salvage myocardium in patients with co-morbidities, such as diabetes and the metabolic syndrome. We investigated delayed preconditioning in mice models of type II diabetes and the metabolic syndrome and investigated interventions to optimize the preconditioning potential.
Hypoxic preconditioning was induced in C57Bl6-mice (WT), leptin deficient ob/ob (model for type II diabetes) and double knock-out (DKO) mice with combined leptin and LDL-receptor deficiency (model for metabolic syndrome). Twenty-four hours later, 30 min of regional ischemia was followed by 60 min reperfusion. Left ventricular contractility and infarct size were studied. The effect of 12 weeks food restriction or angiotensin-converting enzyme inhibition (ACE-I) on this was investigated. Differences between groups were analyzed for statistical significance by student’s t-test or one-way ANOVA followed by a Fisher’s LSD post hoc test. Factorial ANOVA was used to determine the interaction term between preconditioning and treatments, followed by a Fisher’s LSD post hoc test. Two-way ANOVA was used to determine the relationship between infarct size and contractility (PRSW). A value of p<0.05 was considered significant.
Left ventricular contractility is reduced in ob/ob compared with WT and even further reduced in DKO. ACE-I improved contractility in ob/ob and DKO mice. After ischemia/reperfusion without preconditioning, infarct size was larger in DKO and ob/ob versus WT. Hypoxic preconditioning induced a strong protection in WT and a partial protection in ob/ob mice. The preconditioning potential was lost in DKO. Twelve weeks of food restriction or ACE-I restored the preconditioning potential in DKO and improved it in ob/ob.
Delayed preconditioning is restored by food restriction and ACE-I in case of type II diabetes and the metabolic syndrome.
PMCID: PMC3598767  PMID: 23432808
Myocardial protection; Preconditioning; Ischemia/reperfusion injury; Diabetes mellitus; Metabolic syndrome
2.  Ischemia-Reperfusion Injury Leads to Distinct Temporal Cardiac Remodeling in Normal versus Diabetic Mice 
PLoS ONE  2012;7(2):e30450.
Diabetes is associated with higher incidence of myocardial infarction (MI) and increased propensity for subsequent events post-MI. Here we conducted a temporal analysis of the influence of diabetes on cardiac dysfunction and remodeling after ischemia reperfusion (IR) injury in mice. Diabetes was induced using streptozotocin and IR performed by ligating the left anterior descending coronary artery for 30 min followed by reperfusion for up to 42 days. We first evaluated changes in cardiac function using echocardiography after 24 hours reperfusion and observed IR injury significantly decreased the systolic function, such as ejection fraction, fractional shortening and end systolic left ventricular volume (LVESV) in both control and diabetic mice. The longitudinal systolic and diastolic strain rate were altered after IR, but there were no significant differences between diabetic mice and controls. However, a reduced ability to metabolize glucose was observed in the diabetic animals as determined by PET-CT scanning using 2-deoxy-2-(18F)fluoro-D-glucose. Interestingly, after 24 hours reperfusion diabetic mice showed a reduced infarct size and less apoptosis indicated by TUNEL analysis in heart sections. This may be explained by increased levels of autophagy detected in diabetic mice hearts. Similar increases in IR-induced macrophage infiltration detected by CD68 staining indicated no change in inflammation between control and diabetic mice. Over time, control mice subjected to IR developed mild left ventricular dilation whereas diabetic mice exhibited a decrease in both end diastolic left ventricular volume and LVESV with a decreased intraventricular space and thicker left ventricular wall, indicating concentric hypertrophy. This was associated with marked increases in fibrosis, indicted by Masson trichrome staining, of heart sections in diabetic IR group. In summary, we demonstrate that diabetes principally influences distinct IR-induced chronic changes in cardiac function and remodeling, while a smaller infarct size and elevated levels of autophagy with similar cardiac function are observed in acute phase.
PMCID: PMC3275560  PMID: 22347376
3.  CRYAB and HSPB2 deficiency alters cardiac metabolism and paradoxically confers protection against myocardial ischemia in aging mice 
The abundantly expressed small molecular weight proteins, CRYAB and HSPB2, have been implicated in cardioprotection ex vivo. However, the biological roles of CRYAB/HSPB2 coexpression for either ischemic preconditioning and/or protection in situ remain poorly defined. Wild-type (WT) and age-matched (~5–9 mo) CRYAB/HSPB2 double knockout (DKO) mice were subjected either to 30 min of coronary occlusion and 24 h of reperfusion in situ or preconditioned with a 4-min coronary occlusion/4-min reperfusion × 6, before similar ischemic challenge (ischemic preconditioning). Additionally, WT and DKO mice were subjected to 30 min of global ischemia in isolated hearts ex vivo. All experimental groups were assessed for area at risk and infarct size. Mitochondrial respiration was analyzed in isolated permeabilized cardiac skinned fibers. As a result, DKO mice modestly altered heat shock protein expression. Surprisingly, infarct size in situ was reduced by 35% in hearts of DKO compared with WT mice (38.8 ± 17.9 vs. 59.8 ± 10.6% area at risk, P < 0.05). In DKO mice, ischemic preconditioning was additive to its infarct-sparing phenotype. Similarly, infarct size after ischemia and reperfusion ex vivo was decreased and the production of superoxide and creatine kinase release was decreased in DKO compared with WT mice (P < 0.05). In permeabilized fibers, ADP-stimulated respiration rates were modestly reduced and calcium-dependent ATP synthesis was abrogated in DKO compared with WT mice. In conclusion, contrary to expectation, our findings demonstrate that CRYAB and HSPB2 deficiency induces profound adaptations that are related to 1) a reduction in calcium-dependent metabolism/respiration, including ATP production, and 2) decreased superoxide production during reperfusion. We discuss the implications of these disparate results in the context of phenotypic responses reported for CRYAB/HSPB2-deficient mice to different ischemic challenges.
PMCID: PMC3683980  PMID: 17873008
small molecular weight heat shock protein; cardioprotection; aging; preconditioning; reperfusion injury
4.  PTEN inhibitors cause a negative inotropic and chronotropic effect in mice 
European journal of pharmacology  2010;650(1):298-302.
Inactivation of phosphatase and tensin homologue deleted on chromosome ten (PTEN) decreases cardiac contractility under basal conditions and induces cardioprotection against ischemia-reperfusion injury. However, the pharmacological effect of PTEN inhibitors on cardiac contractility has not been studied before. In the present study, we investigated the hypothesis that PTEN inhibition decreases cardiac contractility in mice. We first exposed isolated mouse hearts to the PTEN inhibitor bpV(phen) (40 μM), the phosphoinositide-3 kinase inhibitor wortmannin (1 μM), and the PTEN-resistant PIP3 analog 3-phosphorothioate-PtdIns(3,4,5)P3 (3-PT-PTP, 0.5 μM) for 10 min. Left ventricular pressure was measured by a Mikro-tip pressure catheter. We then inhibited PTEN in mice by intra-peritoneal injection of VO-OHpic (10 μg/kg) 30 min before ischemia and then exposed them to 30 min of ischemia and 120 min of reperfusion. At the end of the experiments, hearts were isolated for measurement of myocardial infarct size by 1.5 % triphenyltetrazolium chloride. Left ventricular systolic pressure and heart rate were significantly decreased by bpV(phen). Consistent with the result, the maximal rate of left ventricular pressure increase or decrease was significantly decreased by bpV(phen). 3-PT-PIP3 mimicked the effect of bpV(phen), and the opposite effect on cardiac contractility was seen with wortmannin. Moreover, inhibition of PTEN in vivo by VO-OHpic decreased left ventricular systolic pressure and heart rate before ischemia, but resulted in an increase in cardiac functional recovery and a decrease in myocardial infarct size after ischemia-reperfusion. In conclusion, PTEN inhibition causes a negative inotropic and chronotropic effect while inducing cardioprotection against ischemia-reperfusion injury.
PMCID: PMC2997895  PMID: 20951693
PTEN; PI3K; cardiac contractility; reperfusion injury; myocardial infarction
5.  ACE-inhibition, but not weight reduction restores cardiomyocyte response to β-adrenergic stimulation in the metabolic syndrome 
Diabetic cardiomyopathy is characterized by systolic and early diastolic ventricular dysfunction. In the metabolic syndrome (MS), ventricular stiffness is additionally increased in a later stage. It is unknown whether this is related to intrinsic cardiomyocyte dysfunction, extrinsic factors influencing cardiomyocyte contractility and/or cardiac function, or a combination of both. A first aim was to study cardiomyocyte contractility and Ca2+ handling in vitro in a mouse model of MS. A second aim was to investigate whether in vivo hypocaloric diet or ACE-inhibition (ACE-I) improved cardiomyocyte contractility in vitro, contractile reserve and Ca2+ handling.
This study was performed in LDL-receptor (LDLR−/−) and leptin-deficient (ob/ob), double knock-out mice (DKO), featuring obesity, type II diabetes, atherogenic dyslipidemia and hypertension. Single knock-out LDLR−/−, ob/ob and wild type mice were used as controls. Cellular contractility, Ca2+ handling and their response to in vivo treatment with diet or ACE-I were studied in isolated cardiomyocytes at baseline, during β-adrenergic stimulation or increased extracellular Ca2+, using field stimulation and patch-clamp.
In untreated conditions, prolongation of contraction-relaxation cycle and altered Ca2+ handling are observed in MS. Response to increased extracellular Ca2+ and β-adrenergic stimulation is impaired and could not be rescued by weight loss. ACE-I restored impaired response to β-adrenergic stimulation in MS, but not the decreased response to increased extracellular Ca2+.
Cardiomyocyte contractility and β-adrenergic response are impaired in MS, due to alterations in cellular Ca2+ handling. ACE-I, but not weight loss, is able to restore cardiomyocyte response to β-adrenergic stimulation in MS.
PMCID: PMC3729821  PMID: 23848952
Metabolic syndrome; Cardiomyocyte contractility; β-adrenergic stimulation; Hypocaloric diet; ACE-inhibition
6.  Anti-Inflammatory and Cardioprotective Effects of Tadalafil in Diabetic Mice 
PLoS ONE  2012;7(9):e45243.
Insulin resistance impairs nitric oxide (NO) bioavailability and obesity promotes a state of chronic inflammation and damages the vascular endothelium. Phosphodiesterase-5 inhibitors restore NO signaling and may reduce circulating inflammatory markers, and improve metabolic parameters through a number of mechanisms. We hypothesized that daily administration of the PDE-5 inhibitor, tadalafil (TAD) will attenuate inflammation, improve fasting plasma glucose and triglyceride levels, body weight, and reduce infarct size after ischemia/reperfusion injury in obese, diabetic mice.
Twenty leptin receptor null (db/db) mice underwent treatment with TAD (1 mg/Kg) or 10% DMSO for 28 days. Body weight and fasting plasma glucose levels were determined weekly. Upon completion, hearts were isolated and subjected to 30 min global ischemia followed by 60 min reperfusion in a Langendorff model. Plasma samples were taken for cytokine analysis and fasting triglyceride levels. Infarct size was measured using computer morphometry of tetrazolium stained sections. Additionally, ventricular cardiomyocytes were isolated and subjected to 40 min of simulated ischemia and reoxygenation. Necrosis was determined using trypan blue exclusion and LDH release assay and apoptosis was assessed by TUNEL assay after 1 h or 18 h of reoxygenation, respectively.
Treatment with TAD caused a reduction in infarct size in the diabetic heart (23.2±1.5 vs. 47.8±3.7%, p<0.01, n = 6/group), reduced fasting glucose levels (292±31.8 vs. 511±19.3 mg/dL, p<0.001) and fasting triglycerides (43.3±21 vs. 129.7±29 mg/dL, p<0.05) as compared to DMSO, however body weight was not significantly reduced. Circulating tumor necrosis factor-α and interleukin-1β were reduced after treatment compared to control (257±16.51 vs. 402.3±17.26 and 150.8±12.55 vs. 264±31.85 pg/mL, respectively; P<0.001) Isolated cardiomyocytes from TAD-treated mice showed reduced apoptosis and necrosis.
We have provided the first evidence that TAD therapy ameliorates circulating inflammatory cytokines and chemokines in a diabetic animal model while improving fasting glucose levels and reducing infarct size following ischemia-reperfusion injury in the heart.
PMCID: PMC3448606  PMID: 23028874
7.  Novel fusion of GLP-1 with a domain antibody to serum albumin prolongs protection against myocardial ischemia/reperfusion injury in the rat 
Glucagon-like peptide-1 (GLP-1) and its mimetics reduce infarct size in the setting of acute myocardial ischemia/reperfusion (I/R) injury. However, the short serum half-life of GLP-1 and its mimetics may limit their therapeutic use in acute myocardial ischemia. Domain antibodies to serum albumin (AlbudAbs) have been developed to extend the serum half-life of short lived therapeutic proteins, peptides and small molecules. In this study, we compared the effect of a long acting GLP-1 agonist, DPP-IV resistant GLP-1 (7–36, A8G) fused to an AlbudAb (GAlbudAb), with the effect of the GLP-1 mimetic, exendin-4 (short half-life GLP-1 agonist) on infarct size following acute myocardial I/R injury.
Male Sprague–Dawley rats (8-week-old) were treated with vehicle, GAlbudAb or exendin-4. Myocardial ischemia was induced 2 h following the final dose for GAlbudAb and 30 min post the final dose for exendin-4. In a subgroup of animals, the final dose of exendin-4 was administered (1 μg/kg, SC, bid for 2 days) 6 h prior to myocardial ischemia when plasma exendin-4 was at its minimum concentration (Cmin). Myocardial infarct size, area at risk and cardiac function were determined 24 h after myocardial I/R injury.
GAlbudAb and exendin-4 significantly reduced myocardial infarct size by 28% and 23% respectively, compared to vehicle (both p < 0.01 vs. vehicle) after I/R injury. Moreover, both GAlbudAb and exendin-4 markedly improved post-ischemic cardiac contractile function. Body weight loss and reduced food intake consistent with the activation of GLP-1 receptors was observed in all treatment groups. However, exendin-4 failed to reduce infarct size when administered 6 h prior to myocardial ischemia, suggesting continuous activation of the GLP-1 receptors is needed for cardioprotection.
Cardioprotection provided by GAlbudAb, a long acting GLP-1 mimetic, following myocardial I/R injury was comparable in magnitude, but more sustained in duration than that produced by short-acting exendin-4. Very low plasma concentrations of exendin-4 failed to protect the heart from myocardial I/R injury, suggesting that sustained GLP-1 receptor activation plays an important role in providing cardioprotection in the setting of acute myocardial I/R injury. Long-acting GLP-1 agonists such as GAlbudAb may warrant additional evaluation as novel therapeutic agents to reduce myocardial I/R injury during acute coronary syndrome.
PMCID: PMC3852957  PMID: 24125539
Glucagon-like peptide-1 (GLP-1); Exendin-4; Anti-albumin-binding domain antibody; Rat; Cardioprotection
8.  Susceptibility to myocardial ischemia reperfusion injury at early stage of type 1 diabetes in rats 
Large body of evidences accumulated in clinical and epidemiological studies indicate that hearts of diabetic subjects are more sensitive to ischemia reperfusion injury (IRI), which results in a higher rate of mortality at post-operation than that of non-diabetes. However, experimental results are equivocal and point to either increased or decreased susceptibility of the diabetic hearts to IRI, especially at the early stage of the disease. The present study was designed to test the hypothesis that the duration/severity of the indexed ischemia is a major determinant of the vulnerability to myocardial IRI at early stage of diabetes.
Four weeks streptozotocin (STZ)-induced diabetic (D) and non-diabetic (C) Sprague–Dawley rats were randomly assigned to receive 30 or 45 min of left anterior descending artery ligation followed by 2 or 3 hours of reperfusion, respectively. Cardiac function was recorded by using Pressure-Volume (PV) conduction system. Myocardial infarct size was determined with triphenyltetrazolium chloride staining. Plasma Creatine kinase-MB (CK-MB), Lactate dehydrogenase (LDH) release, myocardial nitric oxide(NO) content and nitrotyrosine formation, 15-F2t-Isoprostane and plasma superoxide dismutase (SOD) were measured with colorimetric assays. Cardiomyocyte apoptosis was assessed by TUNEL staining. Myocardial TNFα, Caspase-3, STAT3, Akt, and GSK-3β were determined by Western blotting.
Prolongation of ischemia but not reperfusion from 30 min to 45 min significantly increased infarct size in D compared to C rats (P < 0.05), accompanied with significantly increased plasma CK-MB (P < 0.05). Prolongation of the duration of either ischemia or reperfusion significantly increased plasma LDH release and myocardial 15-F2t-Isoprostane and reduced plasma SOD activity, with concomitant reduction of myocardial NO and increase of nitrotyrosine formation in D relative to C (P < 0.05). Prolongation of ischemia and reperfusion significantly reduced left ventricular ejection fraction and increased the peak rate of pressure, accompanied with increased end systolic pressure in D relative to C rats (P < 0.05) but reduced phosphorylations of myocardial STAT3 at site Ser727 and Akt at site Ser473 as well as GSK-3β at Ser 9 (P < 0.05).
Diabetic hearts, even at early stage of the disease are more sensitive to IRI, and this increased severity of post-ischemic myocardial injury depends more on the duration of ischemia than that of reperfusion.
PMCID: PMC3847499  PMID: 24041262
Ischemia reperfusion injury; Diabetes mellitus; Infarct size
9.  Preconditionin effects of dexmedetomidine on myocardial ischemia/reperfusion injury in rats 
Background: Preconditioning might protect the myocardium against ischemia/ reperfusion injury by reducing infarct size and preventing arrhythmias. Dexmedetomidine (DEX) is a highly selective α2-agonist used for sedoanalgesia in daily anesthetic practice. The cardioprotective effects of DEX on infarct size and on the incidence of arrhythmias observed after regional ischemia/reperfusion injury in vivo have not been reported.
Objective: The aim of this study was to determine whether DEX exhibits a preconditioning effect and reduces infarct size and the incidence and duration of arrhythmias in a regional cardiac ischemia/reperfusion model in rats.
Methods: Adult male Sprague-Dawley rats were anesthetized with sodium thiopental and mechanically ventilated (0.9 mL/100 g at 60 strokes/min) through a cannula inserted into the trachea after tracheotomy. Cardiac ischemia was then produced by ligating the left main coronary artery for 30 minutes, followed by a reperfusion period of 120 minutes. Blood pressure (BP) and heart rate (HR) were monitored and echocardiograms (ECGs) were performed. Arrhythmia was scored based on incidence and duration. The animals were randomly divided into 3 groups. The ischemic preconditioning (IPC) group underwent 5 minutes of ischemia followed by 5 minutes of reperfusion before the 30-minute ischemia/120-minute reperfusion period. In the DEX group, intraperitoneal (IP) DEX 1 mL (100 μg/kg) was administered 30 minutes before the ischemia/ reperfusion period. In the control group, IP saline 1 mL was administered 30 minutes before the ischemia/reperfusion period. After reperfusion, the heart was excised, demarcated with saline and ethanol to identify the occluded and nonoccluded myocardium, and cut into slices ~2 mm thick, that were then stained and placed between 2 glass plates. The risk zone and the infarct zone were compared between groups. The investigator assessing the infarcts was blinded to the study group.
Results: Twenty-one adult (aged 4-6 months) male Sprague-Dawley rats weighing 280 to 360 g were included in the study; 7 rats were assigned to each group. BP, HR, and ECG readings were not significantly different between groups and did not change during the study. Arrythmias occurred during ischemia and reperfusion in all groups. The duration of the arrhythmias was significantly shorter and the arrhythmia score was significantly lower in the IPC group (all, P<0.05), compared with the control group; however, they were not significantly different in the DEX group. During the ischemic period, duration of ventricular tachycardia (VT) and ventricular premature contractions (VPC) in the DEX group was significantly longer than that observed in the IPC group (all, P<0.05). The duration of VPC was also significantly shorter than that observed in the control group (both, P<0.05). Duration of VT during the reperfusion period in the DEX group was significantly longer than that observed in both IPC and control groups (both, P<0.05). The mean (SD) percentage of damage was significantly lower in the IPC group (44.1% [2.0%]) and the DEX group (26.7% [2.0%]) compared with the control group (69.0% [3.0%]; both, P<0.05). The percentage of damage in the DEX group was also significantly lower compared with the IPC group (P<0.05).
Conclusions: This small, experimental in vivo study found that DEX was associated with reduced infarct size in ischemia/reperfusion injury in regional ischemia in this rat model but had no effect on the incidence of arrhythmias. Future studies are needed to clarify these findings.
PMCID: PMC3969917  PMID: 24692794
dexmedetomidine; preconditioning; cardiac ischemia/reperfusion
10.  Impaired cardiac ischemic tolerance in spontaneously hypertensive rats is attenuated by adaptation to chronic and acute stress 
The myocardial response to ischemia may be markedly augmented by risk factors associated with lifestyle, leading to left ventricular hypertrophy – an important contributor to cardiovascular morbidity and mortality. Left ventricular hypertrophy and hypertension impairs heart function, and may negatively affect the outcome of ischemia/reperfusion injury. However, ischemic tolerance may persist in altered hearts of hypertensive animals, and may be modified by short- or long-term adaptation to stressful conditions. This article discusses differences in the effects of long-term adaptation to crowding stress and short-term adaptation to ischemic stress, on ischemic tolerance in altered hearts of hypertensive rats.
Chronic hypertension may have a negative impact on the myocardial response to ischemia. On the other hand, intrinsic ischemic tolerance may persist even in the pathologically altered hearts of hypertensive animals, and may be modified by short- or long-term adaptation to different stressful conditions. The effects of long-term limitation of living space (ie, crowding stress [CS]) and brief ischemia-induced stress on cardiac response to ischemia/reperfusion (I/R) injury are not yet fully characterized in hypertensive subjects. The present study was designed to test the influence of chronic and acute stress on the myocardial response to I/R in spontaneously hypertensive rats (SHR) compared with their effects in normotensive counterparts. In both groups, chronic, eight-week CS was induced by caging five rats per cage in cages designed for two rats (200 cm2/rat), while controls (C) were housed four to a cage in cages designed for six animals (480 cm2/rat). Acute stress was evoked by one cycle of I/R (5 min each, ischemic preconditioning) before sustained I/R in isolated Langendorff-perfused hearts of normotensive and SHR rats. At baseline conditions, the effects of CS were manifested only as a further increase in blood pressure in SHR, and by marked limitation of coronary perfusion in normotensive animals, while no changes in heart mechanical function were observed in any of the groups. Postischemic recovery of contractile function, severity of ventricular arrhythmias and lethal injury (infarction size) were worsened in the hypertrophied hearts of C-SHR compared with normotensive C. However, myo-cardial stunning and reperfusion-induced ventricular arrhythmias were attenuated by CS in SHR, which was different from deterioration of I/R injury in the hearts of normotensive animals. In contrast, ischemic preconditioning conferred an effective protection against I/R in both groups, although the extent of anti-infarct and anti-arrhythmic effects was lower in SHR. Both forms of stress may improve the altered response to ischemia in hypertensive subjects. In contrast to short-term preconditioning stress, chronic psychosocial stress was associated with a higher risk of lethal arrhythmias and contractile failure in normotensive animals exposed to an acute ischemic challenge.
PMCID: PMC3209549  PMID: 22065943
Adaptation; Hypertension; Hypertrophy; Myocardial ischemia; Preconditioning; Social stress
11.  CaM Kinase II mediates maladaptive post-infarct remodeling and pro-inflammatory chemoattractant signaling but not acute myocardial ischemia/reperfusion injury 
EMBO Molecular Medicine  2014;6(10):1231-1245.
CaMKII was suggested to mediate ischemic myocardial injury and adverse cardiac remodeling. Here, we investigated the roles of different CaMKII isoforms and splice variants in ischemia/reperfusion (I/R) injury by the use of new genetic CaMKII mouse models. Although CaMKIIδC was upregulated 1 day after I/R injury, cardiac damage 1 day after I/R was neither affected in CaMKIIδ-deficient mice, CaMKIIδ-deficient mice in which the splice variants CaMKIIδB and C were re-expressed, nor in cardiomyocyte-specific CaMKIIδ/γ double knockout mice (DKO). In contrast, 5 weeks after I/R, DKO mice were protected against extensive scar formation and cardiac dysfunction, which was associated with reduced leukocyte infiltration and attenuated expression of members of the chemokine (C-C motif) ligand family, in particular CCL3 (macrophage inflammatory protein-1α, MIP-1α). Intriguingly, CaMKII was sufficient and required to induce CCL3 expression in isolated cardiomyocytes, indicating a cardiomyocyte autonomous effect. We propose that CaMKII-dependent chemoattractant signaling explains the effects on post-I/R remodeling. Taken together, we demonstrate that CaMKII is not critically involved in acute I/R-induced damage but in the process of post-infarct remodeling and inflammatory processes.
PMCID: PMC4287929  PMID: 25193973
apoptosis; Ca2+/calmodulin-dependent protein kinase II; cardiac remodeling; gene replacement; ischemia/reperfusion injury
12.  Photochemically Altered Air Pollution Mixtures and Contractile Parameters in Isolated Murine Hearts before and after Ischemia 
Environmental Health Perspectives  2013;121(11-12):1344-1348.
Background: The cardiopulmonary effects of the individual criteria air pollutants have been well investigated, but little is known about the cardiopulmonary effects of inhaled multipollutant mixtures that more realistically represent environmental exposures.
Objectives: We assessed the cardiopulmonary effects of exposure to photochemically altered particle-free multipollutant mixtures.
Methods: We exposed mice to filtered air (FA), multipollutant mixtures, or ozone (O3) for 4 hr in a photochemical reaction chamber. Eight hours after exposure, we assessed cardiac responses using a Langendorff preparation in a protocol consisting of 20 min of global ischemia followed by 2 hr of reperfusion. Cardiac function was assessed by measuring the index of left-ventricular developed pressure (LVDP) and contractility (dP/dt) before ischemia. On reperfusion after ischemia, recovery of postischemic LVDP and size of infarct were examined. We used bronchoalveolar lavage (BAL) cell counts to assess lung inflammation.
Results: Exposure to the multipollutant mixtures decreased LVDP, baseline rate of left ventricular contraction (dP/dtmaximum), and baseline rate of left ventricular relaxation (dP/dtminimum) compared with exposure to FA. Exposure to O3 also decreased heart rate and dP/dtminimum. Time to ischemic contracture was prolonged in the multipollutant-mixture group relative to that in the FA group. Mice in the multipollutant-mixture group had better recovery of postischemic LVDP and smaller infarct size. Exposure to multipollutant mixtures and to O3 exposure increased numbers of macrophages in the BAL fluid.
Conclusions: Exposure to photochemically altered urban air pollution appears to affect cardiac mechanics in isolated perfused hearts. Inhalation of acute multipollutant mixtures decreases LVDP and cardiac contractility in isolated non-ischemic murine hearts, prolongs ischemic contracture, increases postischemic recovery of LVDP, and reduces infarct size.
Citation: McIntosh-Kastrinsky R, Diaz-Sanchez D, Sexton KG, Jania CM, Zavala J, Tilley SL, Jaspers I, Gilmour MI, Devlin RB, Cascio WE, Tong H. 2013. Photochemically altered air pollution mixtures and contractile parameters in isolated murine hearts before and after ischemia. Environ Health Perspect 121:1344–1348;
PMCID: PMC3855513  PMID: 24148996
13.  Hydrogen sulfide provides cardioprotection against myocardial/ischemia reperfusion injury in the diabetic state through the activation of the RISK pathway 
Medical Gas Research  2014;4(1):20.
Coronary artery disease remains the principal cause of death in patients with diabetes mellitus. Diabetic mice display exacerbated injury following myocardial ischemia-reperfusion (MI/R) and are resistant to most therapeutic interventions. We have reported that sodium sulfide (Na2S) therapy confers cardioprotection during MI/R in non-diabetic mice. Here we tested the hypothesis that Na2S therapy would limit the extent of myocardial injury following MI/R when administered at the time of reperfusion.
Methods and results
Diabetic mice (db/db, 12 weeks of age) were subjected to transient myocardial ischemia for a period of 30 minutes followed by reperfusion up to 24 hours. Na2S (0.05 to 1 mg/kg) or saline (vehicle) was administered into the left ventricular lumen at the time of reperfusion. Na2S therapy significantly decreased myocardial injury in the db/db diabetic mouse, as evidenced by a reduction in infarct size and circulating troponin-I levels. The reduction in myocardial injury was also associated with a reduction in oxidative stress and a decrease in cleaved caspase-3 expression. In an effort to evaluate the signaling mechanism responsible for the observed cardioprotection, additional groups of mice were sacrificed during early reperfusion. Hearts were excised and processed for Western blot analysis. These studies revealed that Na2S therapy activated the Erk1/2 arm of the Reperfusion Injury Salvage Kinase (RISK) pathway.
These findings provide important information that myocardial Erk1/2 activation by Na2S therapy following MI/R sets into motion events, which ultimately lead to cardioprotection in the setting of diabetes.
PMCID: PMC4269946  PMID: 25525500
Diabetes; Cardioprotection; Hydrogen sulfide; RISK pathway; Myocardial Ischemia-reperfusion injury
14.  TRPV1 gene deletion exacerbates inflammation and atypical cardiac remodeling after myocardial infarction 
Hypertension  2008;53(2):243-250.
The transient receptor potential vanilloid (TRPV1) channels expressed in sensory afferent fibers innervating the heart may be activated by proton or endovanilloids released during myocardial ischemia (MI), leading to angina. Although our previous in vitro data indicate that TRPV1 activation may preserve cardiac function after ischemia-reperfusion (I/R) injury, the underlying mechanisms are largely unknown. To test the hypothesis that TRPV1 modulates inflammatory and early remodeling processes to prevent cardiac functional deterioration after myocardial infarction, TRPV1-null mutant (TRPV1-/-) and wild-type (WT) mice were subjected to left anterior descending coronary ligation or sham operation. The infarct size was greater in TRPV1-/- than in WT mice (P < 0.001) 3 days after MI, and the mortality rate was higher in TRPV1-/- than in WT mice (P < 0.05) 7 days after MI. The levels of plasma cardiac troponin I; cytokines including TNF-α, IL-1β, and IL-6; chemokines including MCP-1 and MIP-2; infiltration of inflammatory cells including neutrophil, macrophage, and myofibroblast; as well as collagen contents were greater in TRPV1-/- than in WT mice (P < 0.05) in the infarct area on day 3 and 7 after MI. Changes in left ventricular (LV) geometry led to increased end-systolic and -diastolic diameters and reduced contractile function in TRPV1-/- compared to WT mice. These data show that TRPV1 gene deletion results in excessive inflammation, disproportional LV remodeling, and deteriorated cardiac function after MI, indicating that TRPV1 may prevent infarct expansion and cardiac injury by inhibiting inflammation and abnormal tissue remodeling.
PMCID: PMC2669745  PMID: 19114647
transient receptor potential vanilloid subtype; myocardial infarction; inflammation; early remodeling, transgenic animal model
15.  Magnetic Resonance Imaging (MRI) for the Assessment of Myocardial Viability 
Executive Summary
In July 2009, the Medical Advisory Secretariat (MAS) began work on Non-Invasive Cardiac Imaging Technologies for the Assessment of Myocardial Viability, an evidence-based review of the literature surrounding different cardiac imaging modalities to ensure that appropriate technologies are accessed by patients undergoing viability assessment. This project came about when the Health Services Branch at the Ministry of Health and Long-Term Care asked MAS to provide an evidentiary platform on effectiveness and cost-effectiveness of noninvasive cardiac imaging modalities.
After an initial review of the strategy and consultation with experts, MAS identified five key non-invasive cardiac imaging technologies that can be used for the assessment of myocardial viability: positron emission tomography, cardiac magnetic resonance imaging, dobutamine echocardiography, and dobutamine echocardiography with contrast, and single photon emission computed tomography.
A 2005 review conducted by MAS determined that positron emission tomography was more sensitivity than dobutamine echocardiography and single photon emission tomography and dominated the other imaging modalities from a cost-effective standpoint. However, there was inadequate evidence to compare positron emission tomography and cardiac magnetic resonance imaging. Thus, this report focuses on this comparison only. For both technologies, an economic analysis was also completed.
A summary decision analytic model was then developed to encapsulate the data from each of these reports (available on the OHTAC and MAS website).
The Non-Invasive Cardiac Imaging Technologies for the Assessment of Myocardial Viability is made up of the following reports, which can be publicly accessed at the MAS website at: or at
Positron Emission Tomography for the Assessment of Myocardial Viability: An Evidence-Based Analysis
Magnetic Resonance Imaging for the Assessment of Myocardial Viability: An Evidence-Based Analysis
The objective of this analysis is to assess the effectiveness and cost-effectiveness of cardiovascular magnetic resonance imaging (cardiac MRI) for the assessment of myocardial viability. To evaluate the effectiveness of cardiac MRI viability imaging, the following outcomes were examined: the diagnostic accuracy in predicting functional recovery and the impact of cardiac MRI viability imaging on prognosis (mortality and other patient outcomes).
Clinical Need: Condition and Target Population
Left Ventricular Systolic Dysfunction and Heart Failure
Heart failure is a complex syndrome characterized by the heart’s inability to maintain adequate blood circulation through the body leading to multiorgan abnormalities and, eventually, death. Patients with heart failure experience poor functional capacity, decreased quality of life, and increased risk of morbidity and mortality.
In 2005, more than 71,000 Canadians died from cardiovascular disease, of which, 54% were due to ischemic heart disease. Left ventricular (LV) systolic dysfunction due to coronary artery disease (CAD) 1 is the primary cause of heart failure accounting for more than 70% of cases. The prevalence of heart failure was estimated at one percent of the Canadian population in 1989. Since then, the increase in the older population has undoubtedly resulted in a substantial increase in cases. Heart failure is associated with a poor prognosis: one-year mortality rates were 32.9% and 31.1% for men and women, respectively in Ontario between 1996 and 1997.
Treatment Options
In general, there are three options for the treatment of heart failure: medical treatment, heart transplantation, and revascularization for those with CAD as the underlying cause. Concerning medical treatment, despite recent advances, mortality remains high among treated patients, while, heart transplantation is affected by the limited availability of donor hearts and consequently has long waiting lists. The third option, revascularization, is used to restore the flow of blood to the heart via coronary artery bypass grafting (CABG) or, in some cases, through minimally invasive percutaneous coronary interventions (balloon angioplasty and stenting). Both methods, however, are associated with important perioperative risks including mortality, so it is essential to properly select patients for this procedure.
Myocardial Viability
Left ventricular dysfunction may be permanent, due to the formation of myocardial scar, or it may be reversible after revascularization. Reversible LV dysfunction occurs when the myocardium is viable but dysfunctional (reduced contractility). Since only patients with dysfunctional but viable myocardium benefit from revascularization, the identification and quantification of the extent of myocardial viability is an important part of the work-up of patients with heart failure when determining the most appropriate treatment path. Various non-invasive cardiac imaging modalities can be used to assess patients in whom determination of viability is an important clinical issue, specifically:
dobutamine echocardiography (echo),
stress echo with contrast,
SPECT using either technetium or thallium,
cardiac magnetic resonance imaging (cardiac MRI), and
positron emission tomography (PET).
Dobutamine Echocardiography
Stress echocardiography can be used to detect viable myocardium. During the infusion of low dose dobutamine (5 – 10 µg/kg/min), an improvement of contractility in hypokinetic and akentic segments is indicative of the presence of viable myocardium. Alternatively, a low-high dose dobutamine protocol can be used in which a biphasic response characterized by improved contractile function during the low-dose infusion followed by a deterioration in contractility due to stress induced ischemia during the high dose dobutamine infusion (dobutamine dose up to 40 ug/kg/min) represents viable tissue. Newer techniques including echocardiography using contrast agents, harmonic imaging, and power doppler imaging may help to improve the diagnostic accuracy of echocardiographic assessment of myocardial viability.
Stress Echocardiography with Contrast
Intravenous contrast agents, which are high molecular weight inert gas microbubbles that act like red blood cells in the vascular space, can be used during echocardiography to assess myocardial viability. These agents allow for the assessment of myocardial blood flow (perfusion) and contractile function (as described above), as well as the simultaneous assessment of perfusion to make it possible to distinguish between stunned and hibernating myocardium.
SPECT can be performed using thallium-201 (Tl-201), a potassium analogue, or technetium-99 m labelled tracers. When Tl-201 is injected intravenously into a patient, it is taken up by the myocardial cells through regional perfusion, and Tl-201 is retained in the cell due to sodium/potassium ATPase pumps in the myocyte membrane. The stress-redistribution-reinjection protocol involves three sets of images. The first two image sets (taken immediately after stress and then three to four hours after stress) identify perfusion defects that may represent scar tissue or viable tissue that is severely hypoperfused. The third set of images is taken a few minutes after the re-injection of Tl-201 and after the second set of images is completed. These re-injection images identify viable tissue if the defects exhibit significant fill-in (> 10% increase in tracer uptake) on the re-injection images.
The other common Tl-201 viability imaging protocol, rest-redistribution, involves SPECT imaging performed at rest five minutes after Tl-201 is injected and again three to four hours later. Viable tissue is identified if the delayed images exhibit significant fill-in of defects identified in the initial scans (> 10% increase in uptake) or if defects are fixed but the tracer activity is greater than 50%.
There are two technetium-99 m tracers: sestamibi (MIBI) and tetrofosmin. The uptake and retention of these tracers is dependent on regional perfusion and the integrity of cellular membranes. Viability is assessed using one set of images at rest and is defined by segments with tracer activity greater than 50%.
Cardiac Positron Emission Tomography
Positron emission tomography (PET) is a nuclear medicine technique used to image tissues based on the distinct ways in which normal and abnormal tissues metabolize positron-emitting radionuclides. Radionuclides are radioactive analogs of common physiological substrates such as sugars, amino acids, and free fatty acids that are used by the body. The only licensed radionuclide used in PET imaging for viability assessment is F-18 fluorodeoxyglucose (FDG).
During a PET scan, the radionuclides are injected into the body and as they decay, they emit positively charged particles (positrons) that travel several millimetres into tissue and collide with orbiting electrons. This collision results in annihilation where the combined mass of the positron and electron is converted into energy in the form of two 511 keV gamma rays, which are then emitted in opposite directions (180 degrees) and captured by an external array of detector elements in the PET gantry. Computer software is then used to convert the radiation emission into images. The system is set up so that it only detects coincident gamma rays that arrive at the detectors within a predefined temporal window, while single photons arriving without a pair or outside the temporal window do not active the detector. This allows for increased spatial and contrast resolution.
Cardiac Magnetic Resonance Imaging
Cardiac magnetic resonance imaging (cardiac MRI) is a non-invasive, x-ray free technique that uses a powerful magnetic field, radio frequency pulses, and a computer to produce detailed images of the structure and function of the heart. Two types of cardiac MRI are used to assess myocardial viability: dobutamine stress magnetic resonance imaging (DSMR) and delayed contrast-enhanced cardiac MRI (DE-MRI). DE-MRI, the most commonly used technique in Ontario, uses gadolinium-based contrast agents to define the transmural extent of scar, which can be visualized based on the intensity of the image. Hyper-enhanced regions correspond to irreversibly damaged myocardium. As the extent of hyper-enhancement increases, the amount of scar increases, so there is a lower the likelihood of functional recovery.
Evidence-Based Analysis
Research Questions
What is the diagnostic accuracy of cardiac MRI for detecting myocardial viability?
What is the impact of cardiac MRI viability imaging on prognosis (mortality and other clinical outcomes)?
How does cardiac MRI compare with cardiac PET imaging for the assessment of myocardial viability?
What is the contribution of cardiac MRI viability imaging to treatment decision making?
Is cardiac MRI cost-effective compared with other cardiac imaging modalities for the assessment of myocardial viability?
Literature Search
A literature search was performed on October 9, 2009 using OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, EMBASE, the Cochrane Library, and the International Agency for Health Technology Assessment (INAHTA) for studies published from January 1, 2005 until October 9, 2009. Abstracts were reviewed by a single reviewer and, for those studies meeting the eligibility criteria full-text articles were obtained. In addition, published systematic reviews and health technology assessments were reviewed for relevant studies published before 2005. Reference lists were also examined for any additional relevant studies not identified through the search. The quality of evidence was assessed as high, moderate, low or very low according to GRADE methodology.
Inclusion Criteria
English language full-reports
Published between January 1, 2005 and October 9, 2009
Health technology assessments, systematic reviews, meta-analyses, randomized controlled trials (RCTs), and observational studies
Patients with chronic, known coronary artery disease (CAD)
Used contrast-enhanced MRI
Assessment of functional recovery ≥ 3 months after revascularization
Exclusion Criteria
< 20 patients
< 18 years of age
Patients with non-ischemic heart disease
Studies conducted exclusively in patients with acute myocardial infarction (MI)
Studies where TP, TN, FP, FN cannot be determined
Outcomes of Interest
Positive predictive value (PPV)
Negative Predictive value (NPV)
Positive likelihood ratio
Negative likelihood ratio
Diagnostic accuracy
Mortality rate (for prognostic studies)
Adverse events
Summary of Findings
Based on the available very low quality evidence, MRI is a useful imaging modality for the detection of viable myocardium. The pooled estimates of sensitivity and specificity for the prediction of regional functional recovery as a surrogate for viable myocardium are 84.5% (95% CI: 77.5% – 91.6%) and 71.0% (95% CI: 68.8% – 79.2%), respectively.
Subgroup analysis demonstrated a statistically significant difference in the sensitivity of MRI to assess myocardial viability for studies using ≤25% hyperenhancement as a viability threshold versus studies using ≤50% hyperenhancement as their viability threshold [78.7 (95% CI: 69.1% - 88.2%) and 96.2 (95% CI: 91.8 – 100.6); p=0.0044 respectively]. Marked differences in specificity were observed [73.6 (95% CI: 62.6% - 84.6%) and 47.2 (95% CI: 22.2 – 72.3); p=0.2384 respectively]; however, these findings were not statistically significant.
There were no statistically significant differences between the sensitivities or specificities for any other subgroups including mean preoperative LVEF, imaging method for function recovery assessment, and length of follow-up.
There was no evidence available to determine whether patients with viable myocardium who are revascularized have a lower mortality rate than those who are treated with medical therapy.
PMCID: PMC3426228  PMID: 23074392
16.  Toll-like Receptor 4 Signaling Confers Cardiac Protection Against Ischemic Injury via Inducible Nitric Oxide Synthase- and Soluble Guanylate Cyclase-dependent Mechanisms 
Anesthesiology  2011;114(3):603-613.
Prior administration of a small dose of lipopolysaccharide confers a cardiac protection against ischemia-reperfusion injury. However, the signaling mechanisms that control the protection are incompletely understood. We tested the hypothesis that TLR4 mediates the ability of lipopolysaccharide to protect against cardiac ischemia-reperfusion injury through distinct intracellular pathways involving myeloid differentiation factor 88 (MyD88), TIR-domain-containing adaptor protein inducing interferon-β–mediated transcription-factor (Trif), inducible nitric-oxide synthase (iNOS), and soluble guanylate cyclase (sGC).
Wild-type mice and the genetically modified mice, i.e., TLR4-deficient (TLR4-def), TLR2 knockout (TLR2−/−), MyD88−/−, Trif−/−, iNOS−/−, and sGCα1−/−, were treated with normal saline or 0.1 mg/kg of lipopolysaccharide, intraperitoneally. Twenty-four hours later, isolated hearts were perfused in a Langendorff apparatus and subsequently subjected to 30 min of global ischemia and reperfusion for up to 60 min. Left ventricular function and myocardial infarction sizes were examined.
Compared to saline-treated mice, lipopolysaccharide-treated mice had markedly improved left ventricular developed pressure and dP/dtmax (P < 0.01) and reduced MI sizes (37.2 ± 3.4% vs. 19.8 ± 4.9%, P < 0.01) after ischemia-reperfusion. The cardiac protective effect of lipopolysaccharide was abolished in the TLR4-def and MyD88−/− mice, but remained intact in TLR2−/− or Trif−/− mice. iNOS−/− mice or wild-type mice treated with the iNOS inhibitor 1400W failed to respond to the TLR4-induced nitric oxide production and were not protected by the lipopolysaccharide preconditioning. While sGC 1−/− mice had robust nitric oxide production in response to lipopolysaccharide, they were not protected by the TLR4-elicited cardiac protection.
TLR4 activation confers a potent cardiac protection against ischemia-reperfusion injury via a MyD88-dependent, but Trif-independent mechanism. iNOS/sGC are essential for the TLR4-induced cardiac protection.
PMCID: PMC3044772  PMID: 21270629
17.  Comparison of Cumulative Planimetry versus Manual Dissection to Assess Experimental Infarct Size in Isolated Hearts 
Infarct size (IS) is an important variable to estimate cardiac ischemia/reperfusion injury in animal models. Triphenyltetrazoliumchloride (TTC) stains viable cells red while leaving infarcted cells unstained. To quantify IS, infarcted and non-infarcted tissue is often manually dissected and weighed (IS-DW). An alternative is to measure infarcted areas by cumulative planimetry (IS-CP).
We prospectively compared these two methods in 141 Langendorff-prepared guinea pig hearts (1.44±0.02 g) that were part of different studies on mechanisms of cardioprotection. Hearts were perfused with Krebs-Ringer’s and subjected to 30 min global ischemia after various cardioprotective treatments. Two hours after reperfusion hearts were cut into 6-7 transverse sections (3 mm) and stained for 5 min in 1% TTC and 0.1 M KH2PO4 buffer (pH 7.4, 38°C). Each slice was first scanned and its infarcted area measured with Image 1.62 software (NIH). Infarctions in individual slices of each heart were averaged (IS-CP) on the basis of their weight. After scanning, IS-DW was determined by careful manual dissection of infarcted from noninfarcted tissue and measuring their respective total weight.
We found limited tissue permeation of TTC in relation to the slice thickness leaving tissue in the center unstained, as well as significant cross-contamination of stained vs. unstained tissue after manual dissection. IS-CP and IS-DW ranged from 6.0 to 73.1% and 19.4 to 70.5%, respectively, and correlated as follows: IS-DW = (27.6±1.4) + (0.518±0.038) • IS-CP; r = 0.75 (Pearson), P<0.001. In addition, IS-CP correlated better with return of function after reperfusion like developed left ventricular pressure, contractility and relaxation, and myocardial oxygen consumption.
Despite a good correlation between both methods, limited tissue permeation by TTC diffusion and limited precision in the ability to manually dissect stained from unstained tissue leads to an overestimation of infarct size by dissection and weighing compared to cumulative planimetry.
PMCID: PMC3786703  PMID: 19732842
cumulative planimetry; guinea pig; heart; infarct size; methods; myocardium; planimetry; staining; triphenyltetrazoliumchloride; TTC
18.  Speckle tracking echocardiography in the diagnosis of early left ventricular systolic dysfunction in type II diabetic mice 
The leptin receptor-deficient db/db mouse is a well-established type II diabetes animal model used to investigate diabetic cardiomyopathy. Previous reports have documented diabetic cardiomyopathy is accompanied by cardiac structural and functional abnormalities. To better elucidate early or subtle changes in cardiac performance in db/db mice, we used speckle tracking echocardiography to assess systolic myocardial strain in vivo with diabetic db/db mice in order to study early changes of left ventricle contractile function in type II diabetes model.
Male diabetic db/db mice and age-matched control mice from C57BL/6J strain at 8,12 and 16 weeks of age were subjected to echocardiography. At the midpapillary level in the parasternal left ventricular short-axis view, end diastolic and systolic left ventricular diameter, interventricular septal thickness and posterior wall thicknesses, ejection fraction, fractional shortening were determined by M-mode echocardiography. Using speckle-tracking based strain analysis of two-dimensional echocardiographic images acquired from the parasternal short-axis views at the mid-papillary level, systolic global radial and circumferential strain values were analyzed.
There was no significant difference in interventricular septal thickness, posterior wall thicknesses, end diastolic and systolic left ventricular diameter, ejection fraction and fractional shortening between db/db and age-matched control mice at 8,12 or 16 weeks of age (P > 0.05). At 8 and 12 weeks of age, there was no significant difference in left ventricular radial strain and circumferential strain between db/db mice and age-matched controls (P > 0.05). But at 16 weeks of age, the left ventricular radial strain and circumferential strain in db/db mice were lower than in control mice (P < 0.01).
The present study shows that speckle tracking echocardiography can be used to evaluate cardiac functional alterations in mouse models of cardiovascular disease. Radial and circumferential strain are more sensitive and can be used for detection of early left ventricular contractile dysfunction in db/db type II diabetic mice.
PMCID: PMC4197287  PMID: 25292177
Diabetic cardiomyopathy; db/db mice; Echocardiography; Strain; Cardiac function
19.  Leptin attenuates cardiac apoptosis after chronic ischaemic injury 
Cardiovascular Research  2009;83(2):313-324.
We have previously shown that activation of leptin signalling in the heart reduces cardiac morbidity and mortality after myocardial infarction (MI). In the present study, we tested the hypothesis that leptin signalling limits cardiac apoptosis after MI through activation of signal transducer and activator of transcription (STAT)-3 responsive anti-apoptotic genes, including B-cell lymphoma (bcl)-2 and survivin, that serve to downregulate the activity of caspase-3.
Methods and results
Hearts from C57BL/6J and three groups of leptin-deficient Ob/Ob mice (food-restricted, ad libitum, and leptin-repleted) were examined 4 weeks after permanent left coronary artery ligation or sham operation. Inflammatory and apoptotic cell number was determined in cardiac sections by immunostaining. Expression of cardiac bcl-2, survivin, and pro and active caspase-3 was determined and correlated with in vitro caspase-3 activity. In the absence of MI, both lean and obese leptin-deficient mice exhibited increased cardiac apoptosis compared with wild-type mice. After MI, the highest rates of apoptosis were seen in the infarcted tissue of lean and obese Ob/Ob mice. Further, leptin-deficient hearts, as well as hearts from wild-type mice treated with the STAT-3 inhibitor WP1066, exhibited blunted anti-apoptotic bcl-2 and survivin gene expression, and increased caspase-3 protein expression and activity. The increased caspase-3 activity and apoptosis in hearts of leptin-deficient mice after MI was significantly attenuated in Ob/Ob mice replete with leptin, reducing apoptosis to levels comparable to that observed in wild-type mice after MI.
These results demonstrate that intact leptin signalling post-MI acts through STAT-3 to increase anti-apoptotic bcl-2 and survivin gene expression and reduces caspase-3 activity, consistent with a cardioprotective role of leptin in the setting of chronic ischaemic injury.
PMCID: PMC2701718  PMID: 19233863
Apoptosis; Leptin; Survivin; bcl-2; Heart failure
20.  Postnatal Overfeeding Causes Early Shifts in Gene Expression in the Heart and Long-Term Alterations in Cardiometabolic and Oxidative Parameters 
PLoS ONE  2013;8(2):e56981.
Postnatal overfeeding (OF) in rodents induces a permanent moderate increase in body weight in adulthood. However, the repercussions of postnatal OF on cardiac gene expression, cardiac metabolism and nitro-oxidative stress are less well known.
Methodology/Principal Findings
Immediately after birth, litters of C57BL/6 mice were either maintained at 10 (normal-fed group, NF), or reduced to 3 in order to induce OF. At weaning, mice of both groups received a standard diet. The cardiac gene expression profile was determined at weaning and cardiac metabolism and oxidative stress were assessed at 7 months. The cardiac expression of several genes, including members of the extracellular matrix and apelin pathway, was modified in juvenile OF mice. In adult mice, OF led to an increase in body weight (+30%) and to significant increases in plasma cholesterol, insulin and leptin levels. Myocardial oxidative stress, SOD and catalase activity and mRNA expression were increased in OF mice. In vivo, diastolic and systolic blood pressures were significantly higher and LV shortening and ejection fraction were decreased in OF mice. Ex vivo, after 30 min of ischemia, hearts isolated from OF mice showed lower functional recovery and larger infarct size (31% vs. 54%, p<0.05). Increases in collagen deposition and expression/activity of matrix-metalloproteinase-2 were observed in adult OF mouse hearts. Moreover, an increase in the expression of SOCS-3 and a decrease in STAT-3 phosphorylation were observed in ventricular tissues from OF mice.
Our study emphasizes that over-nutrition during the immediate postnatal period in mice leads to early changes in cardiac gene expression, which may permanently modify the heart’s structural organization and metabolism and could contribute to a greater susceptibility to myocardial ischemia-reperfusion injury.
PMCID: PMC3582632  PMID: 23468899
21.  Broad Suppression of NADPH Oxidase Activity Exacerbates Ischemia/Reperfusion Injury Through Inadvertent Downregulation of HIF-1α and Upregulation of PPARα 
Circulation research  2013;112(8):10.1161/CIRCRESAHA.111.300171.
Nox2 and Nox4 are major components of the NADPH oxidase (Nox) family, which purposefully produce reactive oxidative species (ROS), namely O2− and H2O2, in the heart. The isoform-specific contribution of Nox2 and Nox4 to ischemia/reperfusion (I/R) injury is poorly understood.
We investigated the role of Nox2 and Nox4 in mediating oxidative stress and myocardial injury during I/R using loss of function mouse models.
Methods and Results
Systemic (s) Nox2 KO, sNox4 KO, and cardiac-specific (c) Nox4 KO mice were subjected to I (30 min)/R (24 h). Both myocardial infarct size/area at risk (MI/AAR) and O2− production were lower in sNox2 KO, sNox4 KO, and cNox4 KO than in wild-type (WT) mice. Unexpectedly, however, the MI/AAR was greater, despite less O2− production, in sNox2 KO+cNox4 KO (DKO) mice and transgenic mice with cardiac-specific expression of dominant-negative Nox (Tg-DN-Nox), which suppresses both Nox2 and Nox4, than in WT or single KO mice. Hypoxia-inducible factor-1α (HIF-1α) was downregulated while peroxisome proliferator-activated receptor-alpha (PPARα) was upregulated in Tg-DN-Nox mice. A cross with mice deficient in prolyl hydroxylase 2, which hydroxylates HIF-1α, rescued the I/R injury and prevented upregulation of PPARα in Tg-DN-Nox mice. A cross with PPARα KO mice also attenuated the injury in Tg-DN-Nox mice.
nBoth Nox2 and Nox4 contribute to the increase in ROS and injury by I/R. However, low levels of ROS produced by either Nox2 or Nox4 regulate HIF-1α and PPARα, thereby protecting the heart against I/R, suggesting that Noxs also act as a physiological sensor for myocardial adaptation.
PMCID: PMC3871171  PMID: 23476056
Reactive oxygen species; oxidative stress; free radicals; ischemia/reperfusion
22.  Protease-Activated Receptor-1 Contributes to Cardiac Remodeling and Hypertrophy 
Circulation  2007;116(20):2298-2306.
Protease-activated receptor-1 (PAR-1) is the high-affinity receptor for the coagulation protease thrombin. It is expressed by a variety of cell types in the heart, including cardiomyocytes and cardiac fibroblasts. We have shown that tissue factor (TF) and thrombin contribute to infarct size after cardiac ischemia-reperfusion (I/R) injury. Moreover, in vitro studies have shown that PAR-1 signaling induces hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. The purpose of the present study was to investigate the role of PAR-1 in infarction, cardiac remodeling, and hypertrophy after I/R injury. In addition, we analyzed the effect of overexpression of PAR-1 on cardiomyocytes.
Methods and Results
We found that PAR-1 deficiency reduced dilation of the left ventricle and reduced impairment of left ventricular function 2 weeks after I/R injury. Activation of ERK1/2 was increased in injured PAR-1−/− mice compared with wild-type mice; however, PAR-1 deficiency did not affect infarct size. Cardiomyocyte-specific overexpression of PAR-1 in mice induced eccentric hypertrophy (increased left ventricular dimension and normal left ventricular wall thickness) and dilated cardiomyopathy. Deletion of the TF gene in cardiomyocytes reduced the eccentric hypertrophy in mice overexpressing PAR-1.
Our results demonstrate that PAR-1 contributes to cardiac remodeling and hypertrophy. Moreover, overexpression of PAR-1 on cardiomyocytes induced eccentric hypertrophy. Inhibition of PAR-1 after myocardial infarction may represent a novel therapy to reduce hypertrophy and heart failure in humans.
PMCID: PMC2848478  PMID: 17967980
proteases; receptor, PAR-1; hypertrophy; ventricular remodeling; myocardial infarction
23.  Ranolazine reduces Ca2+ overload and oxidative stress and improves mitochondrial integrity to protect against ischemia reperfusion injury in isolated hearts 
Ranolazine is a clinically approved drug for treating cardiac ventricular dysrhythmias and angina. Its mechanism(s) of protection is not clearly understood but evidence points to blocking the late Na+ current that arises during ischemia, blocking mitochondrial complex I activity, or modulating mitochondrial metabolism. Here we tested the effect of ranolazine treatment before ischemia at the mitochondrial level in intact isolated hearts and in mitochondria isolated from hearts at different times of reperfusion. Left ventricular (LV) pressure (LVP), coronary flow (CF), and O2 metabolism were measured in guinea pig isolated hearts perfused with Krebs-Ringer’s solution; mitochondrial (m) O2•−, Ca2+, NADH/FAD (redox state), and cytosolic (c) Ca2+ were assessed on-line in the LV free wall by fluorescence spectrophotometry. Ranolazine (5 µM), infused for 1 min just before 30 min of global ischemia, itself did not change O2•−, cCa2+, mCa2+ or redox state. During late ischemia and reperfusion (IR) O2•− emission and m[Ca2+] increased less in the ranolazine group vs. the control group. Ranolazine decreased c[Ca2+] only during ischemia while NADH and FAD were not different during IR in the ranolazine vs. control groups. Throughout reperfusion LVP and CF were higher, and ventricular fibrillation was less frequent. Infarct size was smaller in the ranolazine group than the control group. Mitochondria isolated from ranolazine-treated hearts had mild resistance to permeability transition pore (mPTP) opening and less cytochrome c release than control hearts. Ranolazine may provide functional protection of the heart during IR injury by reducing cCa2+ and mCa2+ loading secondary to its effect to block the late Na+ current. Subsequently it indirectly reduces O2•− emission, preserves bioenergetics, delays mPTP opening, and restricts loss of cytochrome c, thereby reducing necrosis and apoptosis.
PMCID: PMC3233383  PMID: 21741479
mitochondrial Ca2+; oxidative stress; permeability transition pore; ranolazine
24.  Impact of the Phosphatidylinositide 3-Kinase Signaling Pathway on the Cardioprotection Induced by Intermittent Hypoxia 
PLoS ONE  2013;8(10):e76659.
Exposure to intermittent hypoxia (IH) may enhance cardiac function and protects heart against ischemia-reperfusion (I/R) injury. To elucidate the underlying mechanisms, we developed a cardioprotective IH model that was characterized at hemodynamic, biochemical and molecular levels.
Mice were exposed to 4 daily IH cycles (each composed of 2-min at 6-8% O2 followed by 3-min reoxygenation for 5 times) for 14 days, with normoxic mice as controls. Mice were then anesthetized and subdivided in various subgroups for analysis of contractility (pressure-volume loop), morphology, biochemistry or resistance to I/R (30-min occlusion of the left anterior descending coronary artery (LAD) followed by reperfusion and measurement of the area at risk and infarct size). In some mice, the phosphatidylinositide 3-kinase (PI3K) inhibitor wortmannin was administered (24 µg/kg ip) 15 min before LAD.
We found that IH did not induce myocardial hypertrophy; rather both contractility and cardiac function improved with greater number of capillaries per unit volume and greater expression of VEGF-R2, but not of VEGF. Besides increasing the phosphorylation of protein kinase B (Akt) and the endothelial isoform of NO synthase with respect to control, IH reduced the infarct size and post-LAD proteins carbonylation, index of oxidative damage. Administration of wortmannin reduced the level of Akt phosphorylation and worsened the infarct size.
We conclude that the PI3K/Akt pathway is crucial for IH-induced cardioprotection and may represent a viable target to reduce myocardial I/R injury.
PMCID: PMC3790757  PMID: 24124584
25.  Cyclic Nucleotide Phosphodiesterase 3A1 Protects the Heart Against Ischemia-Reperfusion Injury 
Journal of molecular and cellular cardiology  2013;64:10.1016/j.yjmcc.2013.08.003.
Phosphodiesterase 3A (PDE3A) is a major regulator of cAMP in cardiomyocytes. PDE3 inhibitors are used for acute treatment of congestive heart failure, but are associated with increased incidence of arrhythmias and sudden death with long-term use. We previously reported that chronic PDE3A downregulation or inhibition induced myocyte apoptosis in vitro. However, the cardiac protective effect of PDE3A has not been demonstrated in vivo in disease models. In this study, we examined the role of PDE3A in regulating myocardial function and survival in vivo using genetically engineered transgenic mice with myocardial overexpression of the PDE3A1 isozyme (TG). TG mice have reduced cardiac function characterized by reduced heart rate and ejection fraction (52.5 ± 7.8% vs. 83.9 ± 4.7%) as well as compensatory expansion of left ventricular diameter (4.19 ± 0.19 mm vs. 3.10 ± 0.18 mm). However, there was no maladaptive increase of fibrosis and apoptosis in TG hearts compared to wild type (WT) hearts, and the survival rates also remained the same. The diminution of cardiac contractile function is very likely attributed to a decrease in beta-adrenergic receptor (β-AR) response in TG mice. Importantly, the myocardial infarct size (4.0 ± 1.8% vs. 24.6 ± 3.8%) and apoptotic cell number (1.3 ± 1.0% vs. 5.6 ± 1.5%) induced by ischemia/reperfusion (I/R) injury were significantly attenuated in TG mice. This was associated with decreased expression of inducible cAMP early repressor (ICER) and increased expression of anti-apoptotic protein BCL-2. To further verify the anti-apoptotic effects of PDE3A1, we performed in vitro apoptosis study in isolated adult TG and WT cardiomyocytes. We found that the apoptotic rates stimulated by hypoxia/reoxygenation or H2O2 were indeed significantly reduced in TG myocytes, and the differences between TG and WT myocytes were completely reversed in the presence of the PDE3 inhibitor milrinone. These together indicate that PDE3A1 negatively regulates β-AR signaling and protects against I/R injury by inhibiting cardiomyocyte apoptosis.
PMCID: PMC3869570  PMID: 23988739
PDE3A; transgenic mice; myocardial injury; myocyte apoptosis; myocyte contractility

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