Glycogen synthase kinase-3beta is a negative regulator of cardiomyocyte hypertrophy.

Hypertrophy is a basic cellular response to a variety of stressors and growth factors, and has been best characterized in myocytes. Pathologic hypertrophy of cardiac myocytes leads to heart failure, a major cause of death and disability in the developed world. Several cytosolic signaling pathways have been identified that transduce prohypertrophic signals, but to date, little work has focused on signaling pathways that might negatively regulate hypertrophy. Herein, we report that glycogen synthase kinase-3beta (GSK-3beta), a protein kinase previously implicated in processes as diverse as development and tumorigenesis, is inactivated by hypertrophic stimuli via a phosphoinositide 3-kinase-dependent protein kinase that phosphorylates GSK-3beta on ser 9. Using adenovirus-mediated gene transfer of GSK-3beta containing a ser 9 to alanine mutation, which prevents inactivation by hypertrophic stimuli, we demonstrate that inactivation of GSK-3beta is required for cardiomyocytes to undergo hypertrophy. Furthermore, our data suggest that GSK-3beta regulates the hypertrophic response, at least in part, by modulating the nuclear/cytoplasmic partitioning of a member of the nuclear factor of activated T cells family of transcription factors. The identification of GSK-3beta as a transducer of antihypertrophic signals suggests that novel therapeutic strategies to treat hypertrophic diseases of the heart could be designed that target components of the GSK-3 pathway.

Developing strategies to link basic cardiovascular sciences with clinical drug development: another opportunity for translational sciences.

Driven, at least in part, by the National Institutes of Health roadmap, an increasing number of studies has bridged the chasm between observations in the basic research laboratory and the clinical bedside. These studies have been an integral part in "translating" new discoveries into therapeutic initiatives. However, "translational medicine" has been used less frequently in the development of cardiovascular drugs or in predicting the potential cardiovascular toxicity of non-cardiac agents. Studies in animal models can provide important clues as to the potential cardiotoxicity of new therapeutic agents, as well as providing a template for the rational design of clinical trials. Three examples of drug development programs that might have been altered by clues available from laboratory studies include the development programs for the anti-cancer drug trastuzumab, the cyclooxygenase inhibitors, and the adenosine-receptor agonists and antagonists. Although mouse models may not always represent the physiology of humans, they provide important information that clinical scientists can utilize in designing safe programs for the evaluation of new pharmacologic agents.

Role of the stress-activated protein kinases in endothelin-induced cardiomyocyte hypertrophy.

The signal transduction pathways governing the hypertrophic response of cardiomyocytes are not well defined. Constitutive activation of the stress-activated protein kinase (SAPK) family of mitogen-activated protein (MAP) kinases or another stress-response MAP kinase, p38, by overexpression of activated mutants of various components of the pathways is sufficient to induce a hypertrophic response in cardiomyocytes, but it is not clear what role these pathways play in the response to physiologically relevant hypertrophic stimuli. To determine the role of the SAPKs in the hypertrophic response, we used adenovirus-mediated gene transfer of SAPK/ERK kinase-1 (KR) [SEK-1(KR)], a dominant inhibitory mutant of SEK-1, the immediate upstream activator of the SAPKs, to block signal transmission down the SAPK pathway in response to the potent hypertrophic agent, endothelin-1 (ET-1). SEK-1(KR) completely inhibited ET-1-induced SAPK activation without affecting activation of the other MAP kinases implicated in the hypertrophic response, p38 and extracellular signal-regulated protein kinases (ERK)-1/ERK-2. Expression of SEK-1(KR) markedly inhibited the ET-1-induced increase in protein synthesis. In contrast, the MAPK/ERK kinase inhibitor, PD98059, which blocks ERK activation, and the p38 inhibitor, SB203580, had no effect on ET-1-induced protein synthesis. ET-1 also induced a significant increase in atrial natriuretic factor mRNA expression as well as in the percentage of cells with highly organized sarcomeres, responses which were also blocked by expression of SEK-1(KR). In summary, inhibiting activation of the SAPK pathway abrogated the hypertrophic response to ET-1. These data are the first demonstration that the SAPKs are necessary for the development of agonist-induced cardiomyocyte hypertrophy, and suggest that in response to ET-1, they transduce critical signals governing the hypertrophic response.

Regulation of cardiac hypertrophy in vivo by the stress-activated protein kinases/c-Jun NH(2)-terminal kinases.

Cardiac hypertrophy often presages the development of heart failure. Numerous cytosolic signaling pathways have been implicated in the hypertrophic response in cardiomyocytes in culture, but their roles in the hypertrophic response to physiologically relevant stimuli in vivo is unclear. We previously reported that adenovirus-mediated gene transfer of SEK-1(KR), a dominant inhibitory mutant of the immediate upstream activator of the stress-activated protein kinases (SAPKs), abrogates the hypertrophic response of neonatal rat cardiomyocytes to endothelin-1 in culture. We now report that gene transfer of SEK-1(KR) to the adult rat heart blocks SAPK activation by pressure overload, demonstrating that the activity of cytosolic signaling pathways can be inhibited by gene transfer of loss-of-function mutants in vivo. Furthermore, gene transfer of SEK-1(KR) inhibited pressure overload-induced cardiac hypertrophy, as determined by echocardiography and several postmortem measures including left ventricular (LV) wall thickness, the ratio of LV weight to body weight, cardiomyocyte diameter, and inhibition of atrial natriuretic factor expression. Our data suggest that the SAPKs are critical regulators of cardiac hypertrophy in vivo, and therefore may serve as novel drug targets in the treatment of hypertrophy and heart failure.

Hsp72-mediated suppression of c-Jun N-terminal kinase is implicated in development of tolerance to caspase-independent cell death.

Pretreatment with mild heat shock is known to protect cells from severe stress (acquired thermotolerance). Here we addressed the mechanism of this phenomenon by using primary human fibroblasts. Severe heat shock (45 degrees C, 75 min) of the fibroblasts caused cell death displaying morphological characteristics of apoptosis; however, it was caspase independent. This cell death process was accompanied by strong activation of Akt, extracellular signal-regulated kinase 1 (ERK1) and ERK2, p38, and c-Jun N-terminal (JNK) kinases. Suppression of Akt or ERK1 and -2 kinases increased cell thermosensitivity. In contrast, suppression of stress kinase JNK rendered cells thermoresistant. Development of thermotolerance was not associated with Akt or ERK1 and -2 regulation, and inhibition of these kinases did not reduce acquired thermotolerance. On the other hand, acquired tolerance to severe heat shock was associated with downregulation of JNK. Using an antisense-RNA approach, we found that accumulation of the heat shock protein Hsp72 is necessary for JNK downregulation and is critical for thermotolerance. The capability of naive cells to withstand moderate heat treatment also appears to be dependent on the accumulation of Hsp72 induced by this stress. Indeed, exposure to 45 degrees C for 45 min caused only transient JNK activation and was nonlethal, while prevention of Hsp72 accumulation prolonged JNK activation and led to massive cell death. We also found that JNK activation by UV irradiation, interleukin-1, or tumor necrosis factor was suppressed in thermotolerant cells and that Hsp72 accumulation was responsible for this effect. Hsp72-mediated suppression of JNK is therefore critical for acquired thermotolerance and may play a role in tolerance to other stresses.

PLIP, a novel splice variant of Tip60, interacts with group IV cytosolic phospholipase A(2), induces apoptosis, and potentiates prostaglandin production.

The group IV cytosolic phospholipase A(2) (cPLA(2)) has been localized to the nucleus (M. R. Sierra-Honigmann, J. R. Bradley, and J. S. Pober, Lab. Investig. 74:684-695, 1996) and is known to translocate from the cytosolic compartment to the nuclear membrane (S. Glover, M. S. de Carvalho, T. Bayburt, M. Jonas, E. Chi, C. C. Leslie, and M. H. Gelb, J. Biol. Chem. 270:15359-15367, 1995; A. R. Schievella, M. K. Regier, W. L. Smith, and L. L. Lin, J. Biol. Chem. 270:30749-30754, 1995). We hypothesized that nuclear proteins interact with cPLA(2) and participate in the functional effects of this translocation. We have identified a nuclear protein, cPLA(2)-interacting protein (PLIP), a splice variant of human Tip60, which interacts with the amino terminal region of cPLA(2). Like Tip60, PLIP cDNA includes the MYST domain containing a C2HC zinc finger and well-conserved similarities to acetyltransferases. Both PLIP and Tip60 coimmunoprecipitate and colocalize with cPLA(2) within the nuclei of transfected COS cells. A polyclonal antibody raised to PLIP recognizes both PLIP and Tip60. Endogenous Tip60 and/or PLIP in rat mesangial cells is localized to the nucleus in response to serum deprivation. Nuclear localization coincides temporally with apoptosis. PLIP expression, mediated by adenoviral gene transfer, potentiates serum deprivation-induced prostaglandin E(2) (PGE(2)) production and apoptosis in mouse mesangial cells from cPLA(2)(+/+) mice but not in mesangial cells derived from cPLA(2)(-/-) mice. Thus PLIP, a splice variant of Tip60, interacts with cPLA(2) and potentiates cPLA(2)-mediated PGE(2) production and apoptosis.

Differential activation of signal transduction pathways in human hearts with hypertrophy versus advanced heart failure.

BACKGROUND: Left ventricular failure is commonly preceded by a period of hypertrophy. Intriguingly, many of the signaling pathways that have been implicated in the regulation of hypertrophy, including the 3 mitogen-activated protein kinases (MAPKs: extracellular signal-regulated kinase, stress-activated protein kinase, and p38), protein phosphatase, calcineurin, and the protein kinase Akt and its target glycogen synthase kinase-3 (GSK-3), also regulate the apoptotic response. METHODS AND RESULTS: To understand the mechanisms that might regulate the progression of heart failure, we analyzed the activity of these signaling pathways in the hearts of patients with advanced heart failure, patients with compensated cardiac hypertrophy, and normal subjects. In patients with hypertrophy, neither the MAPK nor the Akt/GSK-3 pathways were activated, and the dominant signaling pathway was calcineurin. In failing hearts, calcineurin activity was increased but less so than in the hypertrophied hearts, and all 3 MAPKs and Akt were activated (and, accordingly, GSK-3ss was inhibited), irrespective of whether the underlying diagnosis was ischemic or idiopathic cardiomyopathy. CONCLUSIONS: In the failing heart, there is a clear prohypertrophic activity profile, likely occurring in response to increased systolic wall stress and neurohormonal mediators. However, with the activation of these hypertrophic pathways, potent proapoptotic and antiapoptotic signals may also be generated. Therapies directed at altering the balance of activity of these signaling pathways could potentially alter the progression of heart failure.

Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo.

BACKGROUND: The serine-threonine kinase Akt is activated by several ligand-receptor systems previously shown to be cardioprotective. Akt activation reduces cardiomyocyte apoptosis in models of transient ischemia. Its role in cardiac dysfunction or infarction, however, remains unclear. METHODS AND RESULTS: We examined the effects of a constitutively active Akt mutant (myr-Akt) in a rat model of cardiac ischemia-reperfusion injury. In vivo gene transfer of myr-Akt reduced infarct size by 64% and the number of apoptotic cells by 84% (P<0.005 for each). Ischemia-reperfusion injury decreased regional cardiac wall thickening as well as the maximal rate of left ventricular pressure rise and fall (+dP/dt and -dP/dt). Akt activation restored regional wall thickening and +dP/dt and -dP/dt to levels seen in sham-operated rats. To better understand this benefit, we examined the effects of myr-Akt on hypoxic cardiomyocyte dysfunction in vitro. myr-Akt prevented hypoxia-induced abnormalities in cardiomyocyte calcium transients and shortening. Akt activation also enhanced sarcolemmal expression of Glut-4 in vivo and increased glucose uptake in vitro to the level seen with insulin treatment. CONCLUSIONS: Akt activation exerts a powerful cardioprotective effect after transient ischemia that probably reflects its ability to both inhibit cardiomyocyte death and improve function of surviving cardiomyocytes. Akt may represent an important nodal target for therapy in ischemic and other heart disease.

Acute reduction in functional infarct expansion with late coronary reperfusion: assessment with quantitative two-dimensional echocardiography.

Reperfusion performed too late to salvage myocardium decreases chronic infarct expansion in experimental animals. However, the acute effects of delayed reperfusion are not known. Twenty-two dogs underwent 3 (n = 8), 4 (n = 8) or 6 h (n = 6) of circumflex artery occlusion followed by 3 h of reperfusion. Effects of reperfusion on diastolic expansion were assessed in two ways: 1) change in mean radius of curvature of the infarct segment, and 2) change in the ratio of the length of the diameter from the center of the infarct zone to the opposite wall (septal-lateral diameter) to the length of the diameter perpendicular to this (anteroposterior diameter). Effects on systolic expansion were examined with quantitative two-dimensional echocardiographic systolic thickening analysis. Delayed reperfusion produced an immediate decrease in diastolic infarct expansion. The ratio of septal-lateral/anteroposterior diameters, which had increased with occlusion from a preocclusion baseline of 0.98 +/- 0.06 to 1.13 +/- 0.08 (p less than 0.001), decreased with reperfusion to 1.02 +/- 0.07 at 15 min and 1.03 +/- 0.08 at 3 h of reperfusion (p = 0.001). This was due solely to a decrease in the septal-lateral diameter. The radius of curvature of the infarcted segment increased from 2.1 +/- 0.5 cm before reperfusion to 2.74 +/- 0.8 cm at 15 min and 2.6 +/- 0.85 cm at 3 h of reperfusion (p = 0.009). This occurred despite a significant (13.6%) decline in end-diastolic cavity area and is compatible with flattening of the reperfused infarct region. Systolic infarct expansion also improved slightly.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo prediction of the transmural extent of experimental acute myocardial infarction using contrast echocardiography.

Acute myocardial infarction progresses radially from endocardium to epicardium within the ischemic area. The amount of progression is highly variable, but depends largely on the transmural distribution of myocardial blood flow. Recent contrast echocardiographic observations indicate that slowly appearing low levels of contrast enhancement are often seen in the ischemic region, particularly in the epicardial level, and that ischemic regions which show these low levels of contrast have significantly more blood flow than those that do not. This study was designed to determine whether the transmural distribution of this delayed contrast enhancement can sufficiently discriminate between regions of high and low flow to serve as an in vivo predictor of the transmural extent of acute infarction. Twenty-four dogs had acute circumflex coronary ligation which was maintained for 6 hours. Contrast echocardiographic studies were performed at the level of the mitral chordae 2 hours after occlusion using a dilute hydrogen peroxide and blood solution as a contrast agent. Comparison was made with the pathologic infarct measured by triphenyltetrazolium chloride staining. The mean transmural extent of infarction ranged from 0 to 89% and was predicted in vivo by the transmural extent of the delayed contrast defect (r = 0.92; infarction [percent transmural] = 0.74 contrast [percent transmural] + 11%; SEE = 10%). Reproducibility for the transmural extent of delayed contrast defects was good (r = 0.89 to 0.98.) These data further support the concept that the transmural distribution of delayed contrast enhancement parallels blood flow and indicate that the mean transmural extent of acute infarction can be predicted in vivo 2 hours after coronary occlusion from the residual contrast defect.

Exercise testing and ambulatory monitoring in patients with preexcitation syndrome.

Documentation of arrhythmia in symptomatic patients with preexcitation syndromes may be difficult despite extended ambulatory monitoring (MON). We, therefore, examined 12 patients with Wolff-Parkinson-White (WPW) and ten with Lown-Ganong-Levine ECG anomalies to compare the yield of tachyarrhythmia on maximal treadmill testing (MTT) and on MON. Nineteen patients were free of associated heart disease. Average age for the group was 45 years (11 men and 11 women). Sustained atrial arrhythmias developed in four of 22 patients during MTT. All four were free of arrhythmia on subsequent MON. An additional four patients exhibited these arrhythmias on MON. We conclude that the use of both monitoring and exercise testing will enhance detection of arrhythmia in patients with symptomatic preexcitation.

Physiological, pharmacological and toxicological considerations of drug-induced structural cardiac injury.

The incidence of drug-induced structural cardiotoxicity, which may lead to heart failure, has been recognized in association with the use of anthracycline anti-cancer drugs for many years, but has also been shown to occur following treatment with the new generation of targeted anti-cancer agents that inhibit one or more receptor or non-receptor tyrosine kinases, serine/threonine kinases as well as several classes of non-oncology agents. A workshop organized by the Medical Research Council Centre for Drug Safety Science (University of Liverpool) on 5 September 2013 and attended by industry, academia and regulatory representatives, was designed to gain a better understanding of the gaps in the field of structural cardiotoxicity that can be addressed through collaborative efforts. Specific recommendations from the workshop for future collaborative activities included: greater efforts to identify predictive (i) preclinical; and (ii) clinical biomarkers of early cardiovascular injury; (iii) improved understanding of comparative physiology/pathophysiology and the clinical predictivity of current preclinical in vivo models; (iv) the identification and use of a set of cardiotoxic reference compounds for comparative profiling in improved animal and human cellular models; (v) more sharing of data (through publication/consortia arrangements) on target-related toxicities; (vi) strategies to develop cardio-protective agents; and (vii) closer interactions between preclinical scientists and clinicians to help ensure best translational efforts.

Growth factors and mitogen-activated protein kinases.

Mammalian cells respond to external stimuli by activation of a variety of signal transduction pathways, which culminate in stereotypical responses, such as proliferation, growth arrest, hypertrophy, differentiation, or apoptosis. In vertebrates the actions of many stimuli resulting in proliferative or hypertrophic growth converge on a set of cellular kinase cascades, which are collectively called the mitogen-activated protein (MAP) kinase cascades. These MAP kinases have been implicated in vascular smooth muscle cell proliferation and hypertrophy, responses that are central to the pathophysiology of hypertension. In this review, we will examine how proliferative and hypertrophic stimuli activate these MAP kinase cascades, what are the consequences of that activation on gene expression, and how do these signals drive the cell into one of the stereotypical responses noted above.

HSP72 can protect cells from heat-induced apoptosis by accelerating the inactivation of stress kinase JNK.

The major heat shock protein Hsp72 prevents heat-induced apoptosis. We have previously demonstrated that transiently expressed Hsp72 exerts its anti-apoptotic effect by suppressing the activity of stress-kinase JNK, an early component of the apoptotic pathway initiated by heat shock. On the other hand, constitutive expression of Hsp72 does not lead to suppression of heat-induced JNK activation, yet still efficiently prevents apoptosis. To address this apparent contradiction, we studied the effects of constitutively expressed Hsp72 on activation of JNK and apoptosis in Rat-1 fibroblasts. We found that the level of heat-induced apoptosis directly correlated with the duration rather than the magnitude of JNK activity following heat shock. Constitutively expressed Hsp72 strongly reduced the duration of JNK while it did not suppress initial JNK activation. These effects were due to Hsp72-mediated acceleration of JNK dephosphorylation. Addition of vanadate to inhibit JNK phosphatase activity completely prevented the anti-apoptotic action of Hsp72. Therefore, suppression of heat-induced apoptosis by Hsp72 could be fully accounted for by its effects on JNK activity.

Coronary ostial stenosis following aortic valve replacement without continuous coronary perfusion.

The development of coronary ostial stenosis following aortic valve replacement has been attributed to intraoperative trauma to the coronary vessels during continuous coronary perfusion. We describe two patients with this lesion in whom continuous coronary perfusion was not used during aortic valve replacement. Both patients were successfully treated with saphenous vein bypass grafting. Intraoperative observation of the aortic root at the time of the bypass operation in one case revealed the left coronary ostium to be pinpoint in size and involved in a dense fibrous reaction extending up from the sewing ring of the prosthesis. The findings in these cases support the hypothesis that coronary ostial stenosis following aortic valve replacement may be due to a fibrous reaction in the aortic root secondary to turbulent flow through the aortic prosthesis.

Topographic correspondence of contrast echocardiographic perfusion mapping and myocardial infarct extent after varying durations of coronary occlusion.

After acute coronary occlusion, the extent of dysfunction exceeds the extent of infarction by a variable amount. Contrast echocardiography has been shown to be a good predictor of the extent of acute infarction after permanent occlusion. We used hydrogen peroxide contrast echocardiography to study the temporal and topographic relationship between contrast enhancement and tissue viability during acute myocardial infarction in 32 dogs undergoing 1, 2, 3, or 4 hours of circumflex occlusion before reperfusion. To account for changes in collateral blood flow, contrast studies were performed by aortic root injection immediately before reperfusion. The area, circumference, and transmural extent of the region at risk in vivo by contrast echocardiography were statistically unchanged regardless of the duration of occlusion before reperfusion. Echo contrast defect analysis of the risk region predicted the area, circumference, and transmural extent of infarcts reperfused at 2 or more hours (r = 0.81, 0.84, 0.71, respectively). For the 1-hour occlusion group, contrast defect analysis predicted the circumference at risk but markedly overestimated the area and transmural extent of infarction. These data indicate that the circumferential extent of infarction can be identified by contrast echo and is fixed by 1 hour of occlusion. Infarction progression transmurally within the circumferential boundaries had nearly reached the transmural contrast extent by 2 hours of occlusion in this model. Assuming the development of a similar high contrast agent safe for human injection, aortic root contrast echocardiography could be useful to predict myocardium at risk of infarction early after occlusion. Late after occlusion it could be of value to predict the presence of still viable myocardial layers within the dysfunctional infarct region.

Signaling pathways mediating the response to hypertrophic stress in the heart.

Cardiac hypertrophy is an increase in the mass of the heart. It is a major risk factor for the development of myocardial infarction and congestive heart failure, diseases that afflict millions of patients worldwide. Hypertrophy can be caused by intrinsic defects of the proteins of the contractile apparatus of the heart, or by extrinsic stimuli such as hypertension. In this review, we will focus on the cytosolic signal transduction pathways that mediate the hypertrophic response to extrinsic stimuli. Although a large number of signaling molecules have been implicated in the hypertrophic response, we will review data that, we believe, suggest there may be only a few molecules that serve as signaling funnels through which many hypertrophic signals must pass on their way to the nucleus. These include the stress response protein kinases (the stress-activated protein kinases or SAPKs, and, possibly, the p38 kinases) and calcineurin. These molecules have as their primary targets transcription factors, many of which have been implicated in the complex yet stereotypic genetic response to hypertrophic stress. In most cases, it is not possible at present to complete the link from hypertrophic stimulus through a specific signaling molecule and a specific transcription factor to the induction of a specific gene that initiates a particular biologic response. We will attempt to identify some of the most important areas where major questions remain in the hopes of stimulating further research into this major cause of death and disability.

Acute respiratory distress syndrome. Patient position and motion strategies.

A review of the literature since 1974 indicates that, although there is no consensus as to the mechanism, positioning strategies in patients with ARDS improve PaO2 and may diminish lung parenchymal damage. The effect of these maneuvers on patient outcomes is uncertain. There is no uniform algorithm for the application of these techniques; however, there is sufficient evidence cited in the referenced articles to support the use and early application of the techniques to improve oxygenation.