GDF15 Is an Inflammation-Induced Central Mediator of Tissue Tolerance.

Growth and differentiation factor 15 (GDF15) is an inflammation-associated hormone with poorly defined biology. Here, we investigated the role of GDF15 in bacterial and viral infections. We found that inflammation induced GDF15, and that GDF15 was necessary for surviving both bacterial and viral infections, as well as sepsis. The protective effects of GDF15 were largely independent of pathogen control or the magnitude of inflammatory response, suggesting a role in disease tolerance. Indeed, we found that GDF15 was required for hepatic sympathetic outflow and triglyceride metabolism. Failure to defend the lower limit of plasma triglyceride levels was associated with impaired cardiac function and maintenance of body temperature, effects that could be rescued by exogenous administration of lipids. Together, we show that GDF15 coordinates tolerance to inflammatory damage through regulation of triglyceride metabolism.

Mitochondrial network remodeling of the diabetic heart: implications to ischemia related cardiac dysfunction.

Mitochondria play a central role in cellular energy metabolism, and their dysfunction is increasingly recognized as a critical factor in the pathogenesis of diabetes-related cardiac pathophysiology, including vulnerability to ischemic events that culminate in myocardial infarction on the one hand and ventricular arrhythmias on the other. In diabetes, hyperglycemia and altered metabolic substrates lead to excessive production of reactive oxygen species (ROS) by mitochondria, initiating a cascade of oxidative stress that damages mitochondrial DNA, proteins, and lipids. This mitochondrial injury compromises the efficiency of oxidative phosphorylation, leading to impaired ATP production. The resulting energy deficit and oxidative damage contribute to functional abnormalities in cardiac cells, placing the heart at an increased risk of electromechanical dysfunction and irreversible cell death in response to ischemic insults. While cardiac mitochondria are often considered to be relatively autonomous entities in their capacity to produce energy and ROS, their highly dynamic nature within an elaborate network of closely-coupled organelles that occupies 30-40% of the cardiomyocyte volume is fundamental to their ability to exert intricate regulation over global cardiac function. In this article, we review evidence linking the dynamic properties of the mitochondrial network to overall cardiac function and its response to injury. We then highlight select studies linking mitochondrial ultrastructural alterations driven by changes in mitochondrial fission, fusion and mitophagy in promoting cardiac ischemic injury to the diabetic heart.

Muscle LIM Protein Force-Sensing Mediates Sarcomeric Biomechanical Signaling in Human Familial Hypertrophic Cardiomyopathy.

BACKGROUND: Familial hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and is typically caused by mutations in genes encoding sarcomeric proteins that regulate cardiac contractility. HCM manifestations include left ventricular hypertrophy and heart failure, arrythmias, and sudden cardiac death. How dysregulated sarcomeric force production is sensed and leads to pathological remodeling remains poorly understood in HCM, thereby inhibiting the efficient development of new therapeutics. METHODS: Our discovery was based on insights from a severe phenotype of an individual with HCM and a second genetic alteration in a sarcomeric mechanosensing protein. We derived cardiomyocytes from patient-specific induced pluripotent stem cells and developed robust engineered heart tissues by seeding induced pluripotent stem cell-derived cardiomyocytes into a laser-cut scaffold possessing native cardiac fiber alignment to study human cardiac mechanobiology at both the cellular and tissue levels. Coupled with computational modeling for muscle contraction and rescue of disease phenotype by gene editing and pharmacological interventions, we have identified a new mechanotransduction pathway in HCM, shown to be essential in modulating the phenotypic expression of HCM in 5 families bearing distinct sarcomeric mutations. RESULTS: Enhanced actomyosin crossbridge formation caused by sarcomeric mutations in cardiac myosin heavy chain (MYH7) led to increased force generation, which, when coupled with slower twitch relaxation, destabilized the MLP (muscle LIM protein) stretch-sensing complex at the Z-disc. Subsequent reduction in the sarcomeric muscle LIM protein level caused disinhibition of calcineurin-nuclear factor of activated T-cells signaling, which promoted cardiac hypertrophy. We demonstrate that the common muscle LIM protein-W4R variant is an important modifier, exacerbating the phenotypic expression of HCM, but alone may not be a disease-causing mutation. By mitigating enhanced actomyosin crossbridge formation through either genetic or pharmacological means, we alleviated stress at the Z-disc, preventing the development of hypertrophy associated with sarcomeric mutations. CONCLUSIONS: Our studies have uncovered a novel biomechanical mechanism through which dysregulated sarcomeric force production is sensed and leads to pathological signaling, remodeling, and hypertrophic responses. Together, these establish the foundation for developing innovative mechanism-based treatments for HCM that stabilize the Z-disc MLP-mechanosensory complex.

Distinct Roles of Type I and Type III Interferons during a Native Murine ß Coronavirus Lung Infection.

Coronaviruses are a major health care threat to humankind. Currently, the host factors that contribute to limit disease severity in healthy young patients are not well defined. Interferons are key antiviral molecules, especially type I and type III interferons. The role of these interferons during coronavirus disease is a subject of debate. Here, using mice that are deficient in type I (IFNAR1-/-), type III (IFNLR1-/-), or both (IFNAR1/LR1-/-) interferon signaling pathways and murine-adapted coronavirus (MHV-A59) administered through the intranasal route, we define the role of interferons in coronavirus infection. We show that type I interferons play a major role in host survival in this model, while a minimal role of type III interferons was manifested only in the absence of type I interferons or during a lethal dose of coronavirus. IFNAR1-/- and IFNAR1/LR1-/- mice had an uncontrolled viral burden in the airways and lung and increased viral dissemination to other organs. The absence of only type III interferon signaling had no measurable difference in the viral load. The increased viral load in IFNAR1-/- and IFNAR1/LR1-/- mice was associated with increased tissue injury, especially evident in the lung and liver. Type I but not type III interferon treatment was able to promote survival if treated during early disease. Further, we show that type I interferon signaling in macrophages contributes to the beneficial effects during coronavirus infection in mice. IMPORTANCE The antiviral and pathological potential of type I and type III interferons during coronavirus infection remains poorly defined, and opposite findings have been reported. We report that both type I and type III interferons have anticoronaviral activities, but their potency and organ specificity differ. Type I interferon deficiency rendered the mice susceptible to even a sublethal murine coronavirus infection, while the type III interferon deficiency impaired survival only during a lethal infection or during a sublethal infection in the absence of type I interferon signaling. While treatment with both type I and III interferons promoted viral clearance in the airways and lung, only type I interferons promoted the viral clearance in the liver and improved host survival upon early treatment (12 h postinfection). This study demonstrates distinct roles and potency of type I and type III interferons and their therapeutic potential during coronavirus lung infection.

Efficacy of lower doses of pioglitazone after stroke or transient ischaemic attack in patients with insulin resistance.

AIMS: Pioglitazone is a potent insulin-sensitizing drug with anti-atherosclerotic properties, but adverse effects have limited its use. We assessed the benefits and risks of lower versus higher doses of pioglitazone taken by participants in the Insulin Resistance Intervention in Stroke Trial. MATERIALS AND METHODS: Efficacy [myocardial infarction (MI) or recurrent stroke] new-onset diabetes) and adverse outcomes (oedema, weight gain, heart failure and bone fracture) were examined for subjects assigned to pioglitazone or placebo within strata defined by mode dose of study drug taken (i.e. the dose taken on most days in the study). RESULTS: Among the 1938 patients randomized to pioglitazone, the mode dose was <15 mg/day in 546 participants, 15 mg/day in 128, 30 mg/day in 89, and 45 mg/day in 1175. There was no significant effect on stroke/MI or new-onset diabetes with <15 mg/day. For 15 mg/30 mg/day pooled, the adjusted hazard ratios (95% CI) for stroke/MI were 0.48 (0.30, 0.76; p = .002) and 0.74 (0.69, 0.94) for 45 mg/day. For new-onset diabetes, the adjusted hazard ratios were 0.34 (0.15, 0.81; p = .001) and 0.31 (0.59, 0.94; p = .001) respectively. For oedema, weight gain and heart failure, the risk estimates for pioglitazone were lower for subjects taking <45 mg daily. For fractures, the increased risk with pioglitazone was similar across all dose strata. CONCLUSIONS: Lower doses of pioglitazone appear to confer much of the benefit with less adverse effects than the full dose. Further study is needed to confirm these findings so that clinicians may optimize dosing of this secondary prevention strategy in patients with stroke.

Pioglitazone Prevents Stroke in Patients With a Recent Transient Ischemic Attack or Ischemic Stroke: A Planned Secondary Analysis of the IRIS Trial (Insulin Resistance Intervention After Stroke).

BACKGROUND: The IRIS trial (Insulin Resistance Intervention after Stroke) demonstrated that pioglitazone reduced the risk for a composite outcome of stroke or myocardial infarction among nondiabetic patients with insulin resistance and a recent stroke or transient ischemic attack. The current planned secondary analysis uses updated 2013 consensus criteria for ischemic stroke to examine the effect of pioglitazone on stroke outcomes. METHODS: Participants were randomly assigned to receive pioglitazone (45 mg/d target dose) or placebo within 180 days of a qualifying ischemic stroke or transient ischemic attack and were followed for a maximum of 5 years. An independent committee, blinded to treatment assignments, adjudicated all potential stroke outcomes. Time to first stroke event was compared by treatment group, overall and by type of event (ischemic or hemorrhagic), using survival analyses and Cox proportional hazards models. RESULTS: Among 3876 IRIS participants (mean age, 63 years; 65% male), 377 stroke events were observed in 319 participants over a median follow-up of 4.8 years. Pioglitazone was associated with a reduced risk for any stroke at 5 years (8.0% in comparison with 10.7% for the placebo group; hazard ratio [HR], 0.75; 95% confidence interval [CI], 0.60-0.94; log-rank P=0.01). Pioglitazone reduced risk for ischemic strokes (HR, 0.72; 95% CI, 0.57-0.91; P=0.005) but had no effect on risk for hemorrhagic events (HR, 1.00; 95% CI, 0.50-2.00; P=1.00). CONCLUSIONS: Pioglitazone was effective for secondary prevention of ischemic stroke in nondiabetic patients with insulin resistance. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT00091949.

Heart Failure After Ischemic Stroke or Transient Ischemic Attack in Insulin-Resistant Patients Without Diabetes Mellitus Treated With Pioglitazone.

BACKGROUND: The IRIS trial (Insulin Resistance Intervention After Stroke) demonstrated that pioglitazone reduced the risk for both cardiovascular events and diabetes mellitus in insulin-resistant patients. However, concern remains that pioglitazone may increase the risk for heart failure (HF) in susceptible individuals. METHODS: In IRIS, patients with insulin resistance but without diabetes mellitus were randomized to pioglitazone or placebo (1:1) within 180 days of an ischemic stroke or transient ischemic attack and followed for ≤5 years. To identify patients at higher HF risk with pioglitazone, we performed a secondary analysis of IRIS participants without HF history at entry. HF episodes were adjudicated by an external review, and treatment effects were analyzed using time-to-event methods. A baseline HF risk score was constructed from a Cox model estimated using stepwise selection. Baseline patient features (individually and summarized in risk score) and postrandomization events were examined as possible modifiers of the effect of pioglitazone. Net cardiovascular benefit was estimated for the composite of stroke, myocardial infarction, and hospitalized HF. RESULTS: Among 3851 patients, the mean age was 63 years, and 65% were male. The 5-year HF risk did not differ by treatment (4.1% pioglitazone, 4.2% placebo). Risk for hospitalized HF was low and not significantly greater in pioglitazone compared with placebo groups (2.9% versus 2.3%, P=0.36). Older age, atrial fibrillation, hypertension, obesity, edema, high C-reactive protein, and smoking were risk factors for HF. However, the effect of pioglitazone did not differ across levels of baseline HF risk (hazard ratio [95% CI] for pioglitazone versus placebo for patients at low, moderate, and high risk: 1.03 [0.61-1.73], 1.10 [0.56-2.15], and 1.08 [0.58-2.01]; interaction P value=0.98). HF risk was increased in patients with versus those without incident myocardial infarction in both groups (pioglitazone: 31.4% versus 2.7%; placebo: 25.7% versus 2.4%; P<0.0001). Edema, dyspnea, and weight gain in the trial did not predict HF hospitalization but led to more study drug dose reduction with a lower mean dose of pioglitazone versus placebo (29±17 mg versus 33±15 mg, P<0.0001). Pioglitazone reduced the composite outcome of stroke, myocardial infarction, or hospitalized HF (hazard ratio, 0.78; P=0.007). CONCLUSIONS: In IRIS, with surveillance and dose adjustments, pioglitazone did not increase the risk of HF and conferred net cardiovascular benefit in patients with insulin resistance and cerebrovascular disease. The risk of HF with pioglitazone was not modified by baseline HF risk. The IRIS experience may be instructive for maximizing the net benefit of this therapy. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov . Unique identifier: NCT00091949.

Achievement of Guideline-Recommended Weight Loss Among Patients With Ischemic Stroke and Obesity.

Background and Purpose- The proportion of patients with acute ischemic stroke or transient ischemic attack (TIA) and obesity who successfully achieve goals for weight reduction recommended by major professional organizations is unknown. Methods- We examined the experience of participants in the placebo group of the IRIS trial (Insulin Resistance Intervention after Stroke) with a body mass index ≥30 kg/m2 at entry. Patients were of age ≥40 years, with a qualifying stroke or TIA within 180 days of randomization and documented insulin resistance without diabetes mellitus. Weights at baseline and at years 1 and 2 after entry were analyzed to determine the proportion of patients achieving a 5% weight loss and achievement of body mass index <27 kg/m2. Results- Of 1937 subjects assigned to placebo, 855 (44%) had obesity at entry. Median age of these 855 subjects was 60 years (interquartile range, 53-68), 41% were women, and median time from stroke/TIA to trial entry was 79 days. Among 788 subjects in the trial at 1 year, 166 (21%) had lost at least 5% of their starting weight and 12 (2%) had achieved a body mass index <27 kg/m2. One hundred nine (14%) participants gained at least 5% of their baseline weight at 1 year. Among 744 subjects in the trial at 2 years, 185 (25%) had lost at least 5% of their baseline weight and 23 (3%) had achieved a body mass index <27 kg/m2. One hundred forty (19%) participants gained at least 5% of their starting weight at 2 years. Conclusions- Only one quarter of obese patients with a recent ischemic stroke or TIA lost a clinically significant amount of weight after their vascular event. Many patients gained weight. Enhancing weight loss after ischemic stroke or TIA may help improve functional outcome and reduce risk for future vascular events, but clinical trials are needed to test and confirm these potential benefits.

Scoring System to Optimize Pioglitazone Therapy After Stroke Based on Fracture Risk.

Background and Purpose- The insulin sensitizer, pioglitazone, reduces cardiovascular risk in patients after an ischemic stroke or transient ischemic attack but increases bone fracture risk. We conducted a secondary analysis of the IRIS trial (Insulin Resistance Intervention After Stroke) to assess the effect of pretreatment risk for fracture on the net benefits of pioglitazone therapy. Methods- IRIS was a randomized placebo-controlled trial of pioglitazone that enrolled patients with insulin resistance but without diabetes mellitus within 180 days of an ischemic stroke or transient ischemic attack. Participants were recruited at 179 international centers from February 2005 to January 2013 and followed for a median of 4.8 years. Fracture risk models were developed from patient characteristics at entry. Within fracture risk strata, we quantified the effects of pioglitazone compared with placebo by estimating the relative risks and absolute 5-year risk differences for fracture and stroke or myocardial infarction. Results- The fracture risk model included points for age, race-ethnicity, sex, body mass index, disability, and medications. The relative increment in fracture risk with pioglitazone was similar in the lower (

Pioglitazone after Ischemic Stroke or Transient Ischemic Attack.

BACKGROUND: Patients with ischemic stroke or transient ischemic attack (TIA) are at increased risk for future cardiovascular events despite current preventive therapies. The identification of insulin resistance as a risk factor for stroke and myocardial infarction raised the possibility that pioglitazone, which improves insulin sensitivity, might benefit patients with cerebrovascular disease. METHODS: In this multicenter, double-blind trial, we randomly assigned 3876 patients who had had a recent ischemic stroke or TIA to receive either pioglitazone (target dose, 45 mg daily) or placebo. Eligible patients did not have diabetes but were found to have insulin resistance on the basis of a score of more than 3.0 on the homeostasis model assessment of insulin resistance (HOMA-IR) index. The primary outcome was fatal or nonfatal stroke or myocardial infarction. RESULTS: By 4.8 years, a primary outcome had occurred in 175 of 1939 patients (9.0%) in the pioglitazone group and in 228 of 1937 (11.8%) in the placebo group (hazard ratio in the pioglitazone group, 0.76; 95% confidence interval [CI], 0.62 to 0.93; P=0.007). Diabetes developed in 73 patients (3.8%) and 149 patients (7.7%), respectively (hazard ratio, 0.48; 95% CI, 0.33 to 0.69; P<0.001). There was no significant between-group difference in all-cause mortality (hazard ratio, 0.93; 95% CI, 0.73 to 1.17; P=0.52). Pioglitazone was associated with a greater frequency of weight gain exceeding 4.5 kg than was placebo (52.2% vs. 33.7%, P<0.001), edema (35.6% vs. 24.9%, P<0.001), and bone fracture requiring surgery or hospitalization (5.1% vs. 3.2%, P=0.003). CONCLUSIONS: In this trial involving patients without diabetes who had insulin resistance along with a recent history of ischemic stroke or TIA, the risk of stroke or myocardial infarction was lower among patients who received pioglitazone than among those who received placebo. Pioglitazone was also associated with a lower risk of diabetes but with higher risks of weight gain, edema, and fracture. (Funded by the National Institute of Neurological Disorders and Stroke; ClinicalTrials.gov number, NCT00091949.).

AMP-activated protein kinase and adenosine are both metabolic modulators that regulate chloride secretion in the shark rectal gland ( Squalus acanthias).

The production of endogenous adenosine during secretagogue stimulation of CFTR leads to feedback inhibition limiting further chloride secretion in the rectal gland of the dogfish shark (Squalus acanthias). In the present study, we examined the role of AMP-kinase (AMPK) as an energy sensor also modulating chloride secretion through CFTR. We found that glands perfused with forskolin and isobutylmethylxanthine (F + I), potent stimulators of chloride secretion in this ancient model, caused significant phosphorylation of the catalytic subunit Thr172 of AMPK. These findings indicate that AMPK is activated during energy-requiring stimulated chloride secretion. In molecular studies, we confirmed that the activating Thr172 site is indeed present in the α-catalytic subunit of AMPK in this ancient gland, which reveals striking homology to AMPKα subunits sequenced in other vertebrates. When perfused rectal glands stimulated with F + I were subjected to severe hypoxic stress or perfused with pharmacologic inhibitors of metabolism (FCCP or oligomycin), phosphorylation of AMPK Thr172 was further increased and chloride secretion was dramatically diminished. The pharmacologic activation of AMPK with AICAR-inhibited chloride secretion, as measured by short-circuit current, when applied to the apical side of shark rectal gland monolayers in primary culture. These results indicate that that activated AMPK, similar to adenosine, transmits an inhibitory signal from metabolism, that limits chloride secretion in the shark rectal gland.

Cardiac Outcomes After Ischemic Stroke or Transient Ischemic Attack: Effects of Pioglitazone in Patients With Insulin Resistance Without Diabetes Mellitus.

BACKGROUND: Insulin resistance is highly prevalent among patients with atherosclerosis and is associated with an increased risk for myocardial infarction (MI) and stroke. The IRIS trial (Insulin Resistance Intervention after Stroke) demonstrated that pioglitazone decreased the composite risk for fatal or nonfatal stroke and MI in patients with insulin resistance without diabetes mellitus, after a recent ischemic stroke or transient ischemic attack. The type and severity of cardiac events in this population and the impact of pioglitazone on these events have not been described. METHODS: We performed a secondary analysis of the effects of pioglitazone, in comparison with placebo, on acute coronary syndromes (MI and unstable angina) among IRIS participants. All potential acute coronary syndrome episodes were adjudicated in a blinded fashion by an independent clinical events committee. RESULTS: The study cohort was composed of 3876 IRIS participants, mean age 63 years, 65% male, 89% white race, and 12% with a history of coronary artery disease. Over a median follow-up of 4.8 years, there were 225 acute coronary syndrome events, including 141 MIs and 84 episodes of unstable angina. The MIs included 28 (19%) with ST-segment elevation. The majority of MIs were type 1 (94, 65%), followed by type 2 (45, 32%). Serum troponin was 10× to 100× upper limit of normal in 49 (35%) and >100× upper limit of normal in 39 (28%). Pioglitazone reduced the risk of acute coronary syndrome (hazard ratio, 0.71; 95% confidence interval, 0.54-0.94; P=0.02). Pioglitazone also reduced the risk of type 1 MI (hazard ratio, 0.62; 95% confidence interval, 0.40-0.96; log-rank P=0.03), but not type 2 MI (hazard ratio, 1.05; 95% confidence interval, 0.58-1.91; P=0.87). Similarly, pioglitazone reduced the risk of large MIs with serum troponin >100× upper limit of normal (hazard ratio, 0.44; 95% confidence interval, 0.22-0.87; P=0.02), but not smaller MIs. CONCLUSIONS: Among patients with insulin resistance without diabetes mellitus, pioglitazone reduced the risk for acute coronary syndromes after a recent cerebrovascular event. Pioglitazone appeared to have its most prominent effect in preventing spontaneous type 1 MIs. CLINICAL TRIAL REGISTRATION: URL: http://clinicaltrials.gov. Unique identifier: NCT00091949.

Effects of pioglitazone on cognitive function in patients with a recent ischaemic stroke or TIA: a report from the IRIS trial.

INTRODUCTION: Patients with cerebrovascular disease are at increased risk for cognitive dysfunction. Modification of vascular risk factors, including insulin resistance, could improve poststroke cognitive function. METHODS: In the Insulin Resistance Intervention after Stroke (IRIS) trial, patients with a recent ischaemic stroke or transient ischaemic attack (TIA) were randomised to pioglitazone (target 45 mg daily) or placebo. All patients were insulin resistant based on a Homeostasis Model Assessment-Insulin Resistance score >3.0. For this preplanned analysis of cognitive function, we examined the Modified Mini-Mental State Examination (3MS) score (maximum score, 100) during follow-up. Patients were tested at baseline and annually for up to 5 years. Longitudinal mixed model methods were used to compare changes in the 3MS over time. RESULTS: Of the 3876 IRIS participants, 3398 had a 3MS score at baseline and at least once during follow-up and were included in the analysis. Median 3MS score at baseline was 97 (IQR 93-99). The average overall least squared mean 3MS score increased by 0.27 in the pioglitazone group and by 0.29 in the placebo group (mean difference between treatment groups -0.02; 95% CI -0.33 to 0.28, p=0.88). CONCLUSIONS: Among insulin-resistant patients with a recent ischaemic stroke or TIA, pioglitazone did not affect cognitive function, as measured by the 3MS, over 5 years. TRIAL REGISTRATION: ClinicalTrials.gov NCT00091949; Results.

AMPK is critical for mitochondrial function during reperfusion after myocardial ischemia.

AMP-activated kinase (AMPK) is a stress responsive kinase that regulates cellular metabolism and protects against cardiomyocyte injury during ischemia-reperfusion (IR). Mitochondria play an important role in cell survival, but the specific actions of activated AMPK in maintaining mitochondrial integrity and function during reperfusion are unknown. Thus, we assessed the consequences of AMPK inactivation on heart mitochondrial function during reperfusion. Mouse hearts expressing wild type (WT) or kinase-dead (KD) AMPK were studied. Mitochondria isolated from KD hearts during reperfusion had intact membrane integrity, but demonstrated reduced oxidative capacity, increased hydrogen peroxide production and decreased resistance to mitochondrial permeability transition pore opening compared to WT. KD hearts showed increased activation of the mitogen activated protein kinase kinase 4 (MKK4) and downstream c-Jun terminal kinase (JNK) and greater necrosis during reperfusion after coronary occlusion. Transgenic expression of mitochondrial catalase (MCAT) prevented the excessive cardiac JNK activation and attenuated the increased myocardial necrosis observed during reperfusion in KD mice. Inhibition of JNK increased the resistance of KD hearts to mPTP opening, contractile dysfunction and necrosis during IR. Thus, intrinsic activation of AMPK is critical to prevent excess mitochondrial reactive oxygen production and consequent JNK signaling during reperfusion, thereby protecting against mPTP opening, irreversible mitochondrial damage and myocardial injury.

Urocortin 2 autocrine/paracrine and pharmacologic effects to activate AMP-activated protein kinase in the heart.

Urocortin 2 (Ucn2), a peptide of the corticotropin-releasing factor (CRF) family, binds with high affinity to type 2 CRF receptors (CRFR2) on cardiomyocytes and confers protection against ischemia/reperfusion. The mechanisms by which the Ucn2-CRFR2 axis mitigates against ischemia/reperfusion injury remain incompletely delineated. Activation of AMP-activated protein kinase (AMPK) also limits cardiac damage during ischemia/reperfusion. AMPK is classically activated by alterations in cellular energetics; however, hormones, cytokines, and additional autocrine/paracrine factors also modulate its activity. We examined the effects of both the endogenous cardiac Ucn2 autocrine/paracrine pathway and Ucn2 treatment on AMPK regulation. Ucn2 treatment increased AMPK activation and downstream acetyl-CoA carboxylase phosphorylation and glucose uptake in isolated heart muscles. These actions were blocked by the CRFR2 antagonist anti-sauvagine-30 and by a PKCε translocation-inhibitor peptide (εV1-2). Hypoxia-induced AMPK activation was also blunted in heart muscles by preincubation with either anti-sauvagine-30, a neutralizing anti-Ucn2 antibody, or εV1-2. Treatment with Ucn2 in vivo augmented ischemic AMPK activation and reduced myocardial injury and cardiac contractile dysfunction after regional ischemia/reperfusion in mice. Ucn2 also directly activated AMPK in ex vivo-perfused mouse hearts and diminished injury and contractile dysfunction during ischemia/reperfusion. Thus, both Ucn2 treatment and the endogenous cardiac Ucn2 autocrine/paracrine pathway activate AMPK signaling pathway, via a PKCε-dependent mechanism, defining a Ucn2-CRFR2-PKCε-AMPK pathway that mitigates against ischemia/reperfusion injury.

Limiting cardiac ischemic injury by pharmacological augmentation of macrophage migration inhibitory factor-AMP-activated protein kinase signal transduction.

BACKGROUND: Macrophage migration inhibitory factor (MIF) exerts a protective effect on ischemic myocardium by activating AMP-activated protein kinase (AMPK). Small molecules that increase the affinity of MIF for its receptor have been recently designed, and we hypothesized that such agonists may enhance AMPK activation and limit ischemic tissue injury. METHODS AND RESULTS: Treatment of cardiomyocytes with the candidate MIF agonist, MIF20, augmented AMPK phosphorylation, increased by 50% the surface localization of glucose transporter, and enhanced by 25% cellular glucose uptake in comparison with MIF alone. In mouse hearts perfused with MIF20 before no-flow ischemia and reperfusion, postischemic left ventricular function improved commensurately with an increase in cardiac MIF-AMPK activation and an augmentation in myocardial glucose uptake. By contrast, small-molecule MIF agonism was not effective in cells or tissues genetically deficient in MIF or the MIF receptor, verifying the specificity of MIF20 for MIF-dependent AMPK signaling. The protective effect of MIF20 also was evident in an in vivo regional ischemia model. Mice treated with MIF20 followed by left coronary artery occlusion and reperfusion showed a significant reduction in infarcted myocardium. CONCLUSIONS: These data support the pharmacological utility of small-molecule MIF agonists in enhancing AMPK activation and reducing cardiac ischemic injury.

Pioglitazone for secondary prevention after ischemic stroke and transient ischemic attack: rationale and design of the Insulin Resistance Intervention after Stroke Trial.

BACKGROUND: Recurrent vascular events remain a major source of morbidity and mortality after stroke or transient ischemic attack (TIA). The IRIS Trial is evaluating an approach to secondary prevention based on the established association between insulin resistance and increased risk for ischemic vascular events. Specifically, IRIS will test the effectiveness of pioglitazone, an insulin-sensitizing drug of the thiazolidinedione class, for reducing the risk for stroke and myocardial infarction (MI) among insulin resistant, nondiabetic patients with a recent ischemic stroke or TIA. DESIGN: Eligible patients for IRIS must have had insulin resistance defined by a Homeostasis Model Assessment-Insulin Resistance > 3.0 without meeting criteria for diabetes. Within 6 months of the index stroke or TIA, patients were randomly assigned to pioglitazone (titrated from 15 to 45 mg/d) or matching placebo and followed for up to 5 years. The primary outcome is time to stroke or MI. Secondary outcomes include time to stroke alone, acute coronary syndrome, diabetes, cognitive decline, and all-cause mortality. Enrollment of 3,876 participants from 179 sites in 7 countries was completed in January 2013. Participant follow-up will continue until July 2015. SUMMARY: The IRIS Trial will determine whether treatment with pioglitazone improves cardiovascular outcomes of nondiabetic, insulin-resistant patients with stroke or TIA. Results are expected in early 2016.