Nitric oxide has no obligatory role in isoflurane late preconditioning against myocardial stunning.

AIMS: Isoflurane has been demonstrated to produce late preconditioning against myocardial stunning. We tested the hypothesis that this effect is dependent upon an increased production of nitric oxide. MAIN METHODS: Studies were performed in 18 conscious dogs, chronically instrumented to measure coronary blood flow and myocardial wall thickening (WT). In Group 1 (control; n=7), a 10-min coronary occlusion was produced followed by reperfusion; WT was monitored until full recovery. In Group 2 (n=6), the same occlusion-reperfusion protocol was performed 24h after inhalation of 1 MAC isoflurane (1.4% in O(2)) for 60 min. In Group 3 (n=5), the late anti-stunning effect of isoflurane was evaluated following non-selective inhibition of NOS with N-nitro-l-arginine (l-NA, 30 mg/kg on 3 days beginning 1 day prior to isoflurane). Expression of eNOS and iNOS protein was measured by Western blotting. KEY FINDINGS: Two to 3h of reperfusion was required for recovery of WT following isoflurane (Group 2). In contrast, without isoflurane (Group 1), WT remained markedly reduced (30% below baseline) at this time point and required more than 6h of reperfusion for recovery. Treatment with l-NA (Group 3) did not alter time-course of recovery of WT following isoflurane. Isoflurane caused an increased expression of eNOS, but not of iNOS. SIGNIFICANCE: Isoflurane produced late preconditioning against myocardial stunning. Although this effect was associated with an up-regulation of eNOS, its persistence following l-NA suggested that an increased production of nitric oxide did not play an obligatory role.

In vivo gene delivery of XIAP protects against myocardial apoptosis and infarction following ischemia/reperfusion in conscious rabbits.

AIMS: We tested the hypothesis that an in vivo gene delivery of the pro-survival protein XIAP (X-chromosome linked inhibitor of apoptosis protein) protects against myocardial apoptosis and infarction following ischemia/reperfusion. MAIN METHODS: Nineteen rabbits were chronically instrumented with a hydraulic occluder placed around the circumflex coronary artery. Adenovirus harboring XIAP (Ad.XIAP; 1×10(10)pfu/ml) or ß-galactosidase (5×10(9)pfu/ml), as a control, was constructed and transfected into the heart using a catheter placed into the left ventricle accompanied by cross-clamping. 1-2weeks after gene delivery, myocardial ischemia was induced by a 30-min occlusion followed by reperfusion for four days. Protein expression was determined by Western blot and apoptosis (% of myocytes) was quantified by TUNEL staining. KEY FINDINGS: Myocardial infarct size, expressed as a fraction of the area at risk, was reduced in Ad.XIAP (n=5) compared to control (n=7) rabbits (21±3% vs. 30±2%, p<0.05). Apoptosis was reduced in Ad.XIAP rabbits compared to control rabbits (2.96±0.68% vs. 8.98±1.84%, p<0.01). This was associated with an approximate 60% decrease in the cleaved caspase-3 level in Ad.XIAP rabbits compared to control rabbits. SIGNIFICANCE: The current findings demonstrate that overexpression of XIAP via in vivo delivery in an adenovirus can reduce both myocardial apoptosis and infarction following ischemia/reperfusion, at least in part, due to the ability of XIAP to inhibit caspase-3. These findings confirm previous work suggesting a link between myocardial apoptosis and infarction i.e., anti-apoptotic therapy was effective in reducing myocardial infarct size.

Persistency and pathway of isoflurane-induced inhibition of superoxide production by neutrophils.

BACKGROUND: Our previous work has demonstrated that treatment with isoflurane has a preconditioning-like inhibitory effect on superoxide production (SOP) by polymorphonuclear neutrophils. The current objectives were to determine persistency of this effect and to clarify where in the signalling pathway this inhibition of SOP occurred. The latter was accomplished using two receptor-dependent neutrophil agonists, platelet activating factor (PAF) and formyl-methionyl-leucyl-phenylalanine (fMLP), and two receptor-independent neutrophil stimuli, the protein-kinase C stimulator, phorbol myristate acetate (PMA), and the calcium ionophore, A23187. METHODS: Arterial blood samples were obtained from eight dogs under baseline condition (conscious state), during isoflurane (1 MAC) administration, and 24 and 48 hr post-isoflurane (also in conscious state). Neutrophils were isolated and stimulated with 1 muM concentrations of PAF, fMLP, PMA, and A23187. SOP was measured spectrophotometrically. RESULTS: Isoflurane administration caused (1) an approximate 50% decrease in SOP during PAF or fMLP (P < 0.01 vs baseline), which remained evident from 24 to 48 hr following isoflurane; (2) an initial 29% decrease in SOP during PMA (P < 0.05 vs baseline), which returned to baseline by 24 hr following isoflurane; and (3) no change in SOP during A23187 (P > 0.05 vs baseline). CONCLUSIONS: Isoflurane administration caused prolonged (from 24 to 48 hr) decreases in agonist-induced SOP by neutrophils. This effect involved inhibition at site(s) in the signalling pathway upstream from protein kinase C. The current findings suggest that the intraoperative use of isoflurane may result in an extended impairment to the antibacterial host defense mechanism and that neutrophil inhibition may play a role in the delayed tissue protection afforded by treatment with volatile anesthetics.

Sleep-induced hypotension precipitates severe myocardial ischemia.

STUDY OBJECTIVES: Epidemiologic studies have shown a high frequency of major cardiac events at night in patients with coronary artery disease. This has been attributed to the sympathetic surges accompanying rapid eye movement (REM) sleep; the role of non-REM sleep, which comprises 80% of total sleep duration, has been largely neglected. Accordingly, we evaluated the effect of non-REM sleep on contractile function in a region of the left ventricular wall supplied by a flow-limiting coronary stenosis. DESIGN: Eight domestic pigs were chronically instrumented to measure regional left ventricular contractile function (wall thickening), coronary blood flow, and systemic hemodynamic variables. Measurements were obtained: (1) during wakefulness, i.e., conscious condition, prior to imposition of coronary stenosis; (2) during wakefulness following imposition of coronary stenosis (30% reduction of baseline coronary blood flow from 40 +/- 4 to 27 +/- 3 mL/min); and (3) during non-REM sleep with coronary stenosis maintained. RESULTS: During wakefulness, coronary stenosis reduced wall thickening (from 23.3 +/- 3.4% to 15.7 +/- 2.0%), whereas mean arterial pressure and heart rate were unchanged. With coronary stenosis maintained, the onset of non-REM sleep caused 20% decreases in mean arterial pressure and coronary blood flow, accompanied by a cessation of regional wall thickening, i.e., akinesis (wall thickening = 0.2 +/- 2.8%), indicating severe myocardial ischemia. CONCLUSIONS: The arterial hypotension, and associated reduction in coronary blood flow, during non-REM sleep precipitated severe myocardial ischemia in a region of the left ventricular wall supplied by flow-limiting coronary stenosis. Such episodes would occur repeatedly during the sleep cycle and could potentially set the stage for a major cardiac event during the sympathetic activation accompanying REM sleep or morning activities.

Hemodilution does not alter the coronary vasodilating effects of endogenous or exogenous nitric oxide.

INTRODUCTION: It is well known that hemoglobin is a scavenger of nitric oxide (NO). The present study used a canine model to test the hypothesis that acute normovolemic hemodilution (ANH) affects NO-mediated coronary vasodilation. METHODS: Studies were performed in 18 open-chest, anesthetized dogs. In Series 1, the contribution of endogenous NO to coronary vasodilatation during ANH with 5% dextran-40 (reduction in hematocrit by 50%) was assessed. This was accomplished by comparing myocardial blood flow (MBF; radioactive microspheres) in the left anterior descending (LAD) region, which was treated with the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), to that in the circumflex (control) region. In Series 2, the LAD was perfused via a controlled-pressure extracorporeal system with coronary blood flow (CBF) measured with an ultrasonic, transit-time flow transducer. The dose-dependent increases in CBF caused by acetylcholine (ACh), which releases endogenous NO from the vascular endothelium, and sodium nitroprusside (SNP), which provides exogenous NO, were compared before and during ANH. RESULTS: Acute normovolemic hemodilution caused similar (approximately twofold) increases in MBF (P < 0.01) in the absence and presence of L-NAME, and it did not affect the dose-related increases in CBF caused by ACh and SNP. CONCLUSIONS: Series 1: under baseline conditions, hemoglobin in red blood cells does not limit the coronary vasodilatation resulting from tonic release of NO; NO does not mediate coronary vasodilation during ANH. Series 2: ANH does not influence the coronary vasodilating effects of increased levels of NO, whether due to endogenous release (ACh) or infusion of an NO donor (SNP).

Chronic treatment with insulin-like growth factor I enhances myocyte contraction by upregulation of Akt-SERCA2a signaling pathway.

Chronic treatment with insulin-like growth factor I (IGF-I) improves contractile function in congestive heart failure and ischemic cardiomyopathy. The present study investigated the effect of chronic treatment with IGF-I on intrinsic myocyte function and the role of the phosphatidylinositol (PI)3-kinase-Akt-sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a signaling cascade in these responses. Myocytes were isolated from 23 adult rats and cultured with and without IGF-I (10(-6) M). After 48 h of treatment, myocyte function was evaluated. IGF-I increased contractile function (percent contraction, 7.7 +/- 0.3% vs. 4.5 +/- 0.3%; P < 0.01) and accelerated relaxation time (time for 70% relengthening, 81 +/- 4 vs. 106 +/- 5 ms; P < 0.05) compared with untreated myocytes [control (Con)]. The enhanced function was associated with an increase in Ca(2+) transients assessed by fura-2 (340/380 nm; IGF-I, 0.42 +/- 0.02 vs. Con, 0.25 +/- 0.01; P < 0.01). The PI3-kinase inhibitor LY-249002 (10(-9) M) abolished the enhanced function caused by IGF-I. IGF-I increased both Akt and SERCA2a protein levels 2.5- and 4.8-fold, respectively, compared with those of Con (P < 0.01); neither phospholamban nor calsequestrin was affected. To evaluate whether the SERCA2a protein was directly mediated by Akt-SERCA2a signaling, IGF-I-induced changes in the SERCA2a protein were compared in myocytes transfected with adenovirus harboring either constitutively active Akt [multiplicity of infection (MOI), 15] or dominant negative Akt (dnAkt; MOI, 15). The ability of IGF-I to upregulate the SERCA2a protein in myocytes transfected with active Akt was absent in dnAkt myocytes. Taken together, our findings indicate that chronic treatment with IGF-I enhances intrinsic myocyte function and that this effect is due to an enhancement in intracellular Ca(2+) handling, secondary to the activation of the PI3-kinase-Akt-SERCA2a signaling cascade.

Evidence for enhanced eNOS function in coronary microvessels during the second window of protection.

Nitric oxide (NO) derived from endothelial NO synthase (NOS) (eNOS) has been identified as a trigger for the second window of protection (SWOP), but its role as a mediator during the SWOP is a matter of debate. Eighteen mongrel dogs were chronically instrumented to measure left ventricular function, coronary blood flow, and wall thickening. Myocardial preconditioning was induced by 10 min coronary artery occlusion. After 24 h of reperfusion (during the SWOP), the hearts were excised. Coronary microvessels were isolated and incubated in presence of 1) the endothelium-dependent agonists carbachol and bradykinin, 2) the calcium ionophore A23187, and 3) the angiotensin-converting enzyme (ACE) inhibitors enalaprilat and ramiprilat. Nitrite, a metabolite of NO, was measured. Under baseline conditions, nitrite production in microvessels from SWOP was 30% higher than that from normal (96 +/- 4 vs. 74 +/- 3 pmol/mg, P < 0.01, respectively). Nitrite production in response to carbachol, bradykinin, and A23187 was also enhanced in microvessels from SWOP (P < 0.05). These enhanced responses were abolished by N(G)-nitro-l-arginine methyl ester (l-NAME) or the endothelial receptor-specific antagonists atropine and HOE-140. The level of eNOS protein in the SWOP myocardium was twofold higher than that in the non-SWOP myocardium. Nitrite production in response to the ACE inhibitors was greater in microvessels from SWOP. These effects were blocked by l-NAME, HOE-140, or dichloroisocoumarin (which inhibits kinin formation). We found that a brief ischemic episode induced delayed, enhanced NO production in coronary microvessels and an upregulation of eNOS protein. These findings suggest that eNOS is a mediator during the SWOP. The ability of ACE inhibitors to enhance NO release during the SWOP points to an additional clinical application for these drugs.

Autophagy in chronically ischemic myocardium.

We tested the hypothesis that chronically ischemic (IS) myocardium induces autophagy, a cellular degradation process responsible for the turnover of unnecessary or dysfunctional organelles and cytoplasmic proteins, which could protect against the consequences of further ischemia. Chronically instrumented pigs were studied with repetitive myocardial ischemia produced by one, three, or six episodes of 90 min of coronary stenosis (30% reduction in baseline coronary flow followed by reperfusion every 12 h) with the non-IS region as control. In this model, wall thickening in the IS region was chronically depressed by approximately 37%. Using a nonbiased proteomic approach combining 2D gel electrophoresis with in-gel proteolysis, peptide mapping by MS, and sequence database searches for protein identification, we demonstrated increased expression of cathepsin D, a protein known to mediate autophagy. Additional autophagic proteins, cathepsin B, heat shock cognate protein Hsc73 (a key protein marker for chaperone-mediated autophagy), beclin 1 (a mammalian autophagy gene), and the processed form of microtubule-associated protein 1 light chain 3 (a marker for autophagosomes), were also increased. These changes, not evident after one episode, began to appear after two or three episodes and were most marked after six episodes of ischemia, when EM demonstrated autophagic vacuoles in chronically IS myocytes. Conversely, apoptosis, which was most marked after three episodes, decreased strikingly after six episodes, when autophagy had increased. Immunohistochemistry staining for cathepsin B was more intense in areas where apoptosis was absent. Thus, autophagy, triggered by ischemia, could be a homeostatic mechanism, by which apoptosis is inhibited and the deleterious effects of chronic ischemia are limited.

Paradoxical cellular Ca2+ signaling in severe but compensated canine left ventricular hypertrophy.

In conscious dogs with severe left ventricular (LV) hypertrophy (H) (doubling of LV/body weight), which developed gradually over 1 to 2 years after aortic banding, baseline LV function was well compensated. The LV was able to generate twice the LV systolic pressure without an increase in LV end-diastolic pressure, or decrease in LV dP/dt or LV wall thickening. However, LV myocytes isolated from LVH dogs exhibited impaired contraction at baseline and in response to Ca2+. There was no change in L-type Ca2+ channel current (ICa) density but the ability of ICa to trigger Ca2+ release from the sarcoplasmic reticulum (SR) was reduced. Immunoblot analysis revealed a 68% decrease in SERCA2a, and a 35% decrease in the number of ryanodine receptors (RyR2), with no changes in protein level of calsequestrin, Na+/Ca2+ exchanger or phospholamban (PLB), but with both RyR2 and PLB hyperphosphorylated. Spontaneous Ca2+ sparks in LVH cells were found to have prolonged duration but similar intensities despite the reduced SR Ca2+ load. A higher Ca2+ spark rate was observed in LVH cells, but this is inconsistent with the reduced SR Ca2+ content. However, Ca2+ waves were found to be less frequent, slower and were more likely to be aborted in Ca2+-challenged LVH cells. These paradoxical observations could be accounted for by a nonuniform SR Ca2+ distribution, RyR2 hyperphosphorylation in the presence of decreased global SR Ca2+ load. We conclude that severe LVH with compensation masks cellular and subcellular Ca2+ defects that remain likely contributors to the limited contractile reserve of LVH.

Program of cell survival underlying human and experimental hibernating myocardium.

Hibernating myocardium refers to chronically dysfunctional myocardium in patients with coronary artery disease in which cardiac viability is maintained and whose function improves after coronary revascularization. It is our hypothesis that long-term adaptive genomic mechanisms subtend the survival capacity of this ischemic myocardium. Therefore, the goal of this study was to determine whether chronic repetitive ischemia elicits a gene program of survival protecting hibernating myocardium against cell death. Accordingly, we measured the expression of survival genes in hibernating myocardium, both in patients surgically treated for hibernation and in a chronic swine model of repetitive ischemia reproducing the features of hibernation. Human hibernating myocardium was characterized by an upregulation of genes and corresponding proteins involved in anti-apoptosis (IAP), growth (VEGF, H11 kinase), and cytoprotection (HSP70, HIF-1alpha, GLUT1). In the swine model, the same genes and proteins were upregulated after repetitive ischemia, which was accompanied by a concomitant decrease in myocyte apoptosis. These changes characterize viable tissue, because they were not found in irreversibly injured myocardium. Our report demonstrates a novel mechanism by which the activation of an endogenous gene program of cell survival underlies the sustained viability of the hibernating heart. Potentially, promoting such a program offers a novel opportunity to salvage postmitotic tissues in conditions of ischemia.

Computer model of action potential of mouse ventricular myocytes.

We have developed a mathematical model of the mouse ventricular myocyte action potential (AP) from voltage-clamp data of the underlying currents and Ca2+ transients. Wherever possible, we used Markov models to represent the molecular structure and function of ion channels. The model includes detailed intracellular Ca2+ dynamics, with simulations of localized events such as sarcoplasmic Ca2+ release into a small intracellular volume bounded by the sarcolemma and sarcoplasmic reticulum. Transporter-mediated Ca2+ fluxes from the bulk cytosol are closely matched to the experimentally reported values and predict stimulation rate-dependent changes in Ca2+ transients. Our model reproduces the properties of cardiac myocytes from two different regions of the heart: the apex and the septum. The septum has a relatively prolonged AP, which reflects a relatively small contribution from the rapid transient outward K+ current in the septum. The attribution of putative molecular bases for several of the component currents enables our mouse model to be used to simulate the behavior of genetically modified transgenic mice.

Aging increases stiffness of cardiac myocytes measured by atomic force microscopy nanoindentation.

It is well established that the aging heart exhibits left ventricular (LV) diastolic dysfunction and changes in mechanical properties, which are thought to be due to alterations in the extracellular matrix. We tested the hypothesis that the mechanical properties of cardiac myocytes significantly change with aging, which could contribute to the global changes in LV diastolic dysfunction. We used atomic force microscopy (AFM), which determines cellular mechanical property changes at nanoscale resolution in myocytes, from young (4 mo) and old (30 mo) male Fischer 344 x Brown Norway F1 hybrid rats. A measure of stiffness, i.e., apparent elastic modulus, was determined by analyzing the relationship between AFM indentation force and depth with the classical infinitesimal strain theory and by modeling the AFM probe as a blunted conical indenter. This is the first study to demonstrate a significant increase (P < 0.01) in the apparent elastic modulus of single, aging cardiac myocytes (from 35.1 +/- 0.7, n = 53, to 42.5 +/- 1.0 kPa, n = 58), supporting the novel concept that the mechanism mediating LV diastolic dysfunction in aging hearts resides, in part, at the level of the myocyte.

Insights into cardioprotection obtained from study of cellular Ca2+ handling in myocardium of true hibernating mammals.

Mammalian hibernators exhibit remarkable resistance to low body temperature, whereas non-hibernating (NHB) mammals develop ventricular dysfunction and arrhythmias. To investigate this adaptive change, we compared contractile and electrophysiological properties of left ventricular myocytes isolated from hibernating (HB) woodchucks (Marmota monax) and control NHB woodchucks. The major findings of this study were the following: 1) the action potential duration in HB myocytes was significantly shorter than in NHB myocytes, but the amplitude of peak contraction was unchanged; 2) HB myocytes had a 33% decreased L-type Ca2+ current (I(Ca)) density and twofold faster I(Ca) inactivation but no change in the current-voltage relationship; 3) there were no changes in the density of inward rectifier K+ current, transient outward K+ current, or Na+/Ca2+ exchange current, but HB myocytes had increased sarcoplasmic reticulum Ca2+ content as estimated from caffeine-induced Na+/Ca2+ exchange current values; 4) expression of the L-type Ca2+ channel alpha(1C)-subunit was decreased by 30% in HB hearts; and 5) mRNA and protein levels of sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a), phospholamban, and the Na+/Ca2+ exchanger showed a pattern that is consistent with functional measurements: SERCA2a was increased and phospholamban was decreased in HB relative to NHB hearts with no change in the Na+/Ca2+ exchanger. Thus reduced Ca2+ channel density and faster I(Ca) inactivation coupled to enhanced sarcoplasmic reticulum Ca2+ release may underlie shorter action potentials with sustained contractility in HB hearts. These changes may account for natural resistance to Ca2+ overload-related ventricular dysfunction and point to an important cardioprotective mechanism during true hibernation.

Mesenteric lymph from rats with thermal injury prolongs the action potential and increases Ca2+ transient in rat ventricular myocytes.

Although gut-derived mesenteric lymph from animals with thermal injury appears to lead to myocardial contractile dysfunction, the cellular mechanisms remain unclear. We examined the direct effects of intestinal lymph on excitation-contraction coupling in rat ventricular myocytes. Lymph from rats receiving burn injury (burn lymph), but not from sham-burned rats, rapidly enhanced myocyte contraction and the amplitude of Ca2+ transient; the average percentage of shortening was increased from 5.5 +/- 0.3% to 10.5 +/- 0.9%. 90% and the Ca2+ transients increased by 80% +/- 20%. Burn lymph had no effect on the amplitude of L-type Ca2+ current (ICa) or the inward rectifier K+ current, but the transient outward K+ currents (Ito) were reduced significantly by burn lymph. Inhibition of Ito was not altered by an alpha1-adrenergic receptor (AR) antagonist, prazosin, indicating that the block was not mediated via alpha1-AR signaling pathway. Action potential (AP) duration, measured at 50% and 90% repolarization, was prolonged by burn lymph. Stimulation of myocytes with AP voltage-clamp waveforms derived from prolonged AP induced by burn lymph revealed a 1.7-fold increase in Ca2+ influx via ICa compared with the Ca2+ influx induced by control AP. Blocking of Ito by 4-aminopyridine prolonged AP duration and increased Ca2+ transients, mimicking the effects of burn lymph. Burn lymph did not affect Na+/Ca2+ exchange currents or caffeine-induced SR Ca2+ release. Thus, acute exposure of normal cardiac myocytes to burn lymph increases Ca2+ transients by a prolongation of AP as a result of a reduction of Ito with no intrinsic change in ICa or exchanger. The electrophysiological changes are similar to those that occur during compensated cardiac hypertrophy, suggesting a common mechanistic link between burn lymph- and hypertrophy-induced cardiac dysfunction.

Mechanism of enhanced cardiac function in mice with hypertrophy induced by overexpressed Akt.

Transgenic mice with cardiac-specific overexpression of active Akt (TG) not only exhibit hypertrophy but also show enhanced left ventricular (LV) function. In 3-4-month-old TG, heart/body weight was increased by 60% and LV ejection fraction was elevated (84 +/- 2%, p < 0.01) compared with nontransgenic littermates (wild type (WT)) (73 +/- 1%). An increase in isolated ventricular myocyte contractile function (% contraction) in TG compared with WT (6.1 +/- 0.2 versus 3.5 +/- 0.2%, p < 0.01) was associated with increased Fura-2 Ca2+ transients (396 +/- 50 versus 250 +/- 24 nmol/liter, p < 0.05). The rate of relaxation (+dL/dt) was also enhanced in TG (214 +/- 15 versus 98 +/- 18 microm/s, p < 0.01). L-type Ca2+ current (ICa) density was increased in TG compared with WT (-9.0 +/- 0.3 versus 7.2 +/- 0.3 pA/pF, p < 0.01). Sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) protein levels were increased (p < 0.05) by 6.6-fold in TG, which could be recapitulated in vitro by adenovirus-mediated overexpression of Akt in cultured adult ventricular myocytes. Conversely, inhibiting SERCA with either ryanodine or thapsigargin affected myocyte contraction and relaxation and Ca2+ channel kinetics more in TG than in WT. Thus, myocytes from mice with overexpressed Akt demonstrated enhanced contractility and relaxation, Fura-2 Ca2+ transients, and Ca2+ channel currents. Furthermore, increased protein expression of SERCA2a plays an important role in mediating enhanced LV function by Akt. Up-regulation of SERCA2a expression and enhanced LV myocyte contraction and relaxation in Akt-induced hypertrophy is opposite to the down-regulation of SERCA2a and reduced contractile function observed in many other forms of LV hypertrophy.

Neurally-mediated increase in calcineurin activity regulates cardiac contractile function in absence of hypertrophy.

OBJECTIVE: The calcineurin pathway has been involved in the development of cardiac hypertrophy, yet it remains unknown whether calcineurin activity can be regulated in myocardium independently from hypertrophy and cardiac load. METHODS: To test that hypothesis, we measured calcineurin activity in a rat model of infrarenal aortic constriction (IR), which affects neurohormonal pathways without increasing cardiac afterload. RESULTS: In this model, there was no change in arterial pressure over the 4-week experimental period, and the left ventricle/body weight ratio did not increase. At 2 weeks after IR, calcineurin activity was increased 1.8-fold (P<0.05) and remained elevated at 4 weeks (1.7-fold, P<0.05). Similarly, the cardiac activity of calcium calmodulin kinase II (CaMKII) was increased significantly after IR, which confirms a regulation of Ca(2+)-dependent enzymes in this model. In cardiac myocytes, the increased activity of calcineurin was accompanied by a significant decrease in L-type Ca(2+) channel activity (I(Ca)) and contraction velocity (-dL/dt). Cardiac denervation prevented the activation of calcineurin after IR, which demonstrates that a neurohormonal mechanism is responsible for the changes in enzymatic activity. In addition, cardiac denervation suppressed the effects of IR on I(Ca) and -dL/dt, which shows that calcineurin activation is related to altered contractility. However, action potential duration, the densities of inward rectifier K(+) currents (I(K1)), and outward K(+) currents (I(to) and I(K)) were not altered in IR myocytes. CONCLUSIONS: Calcineurin can be activated in the heart through a neural stimulus, which induces alterations in Ca(2+) currents and contractility. These effects occur in the absence of myocyte hypertrophy, electrophysiological changes in action potential, and K(+) channel currents.

Adding angiotensin II type 1 receptor blockade to angiotensin-converting enzyme inhibition limits myocyte remodeling after myocardial infarction.

BACKGROUND: Adding angiotensin II type 1 receptor blockade (ARB) to angiotensin-converting enzyme inhibition (ACEI) further attenuates left ventricular (LV) remodeling in an ovine model of myocardial infarction (MI). We hypothesized that combined therapy with ACEI and ARB (CT) would be additive in the limitation of the myocyte hypertrophy and dysfunction that occurs in untreated adjacent noninfarcted regions during remodeling. METHODS AND RESULTS: Nineteen sheep underwent coronary ligation to create a moderate-sized anteroapical infarction. Post-MI day 2, sheep were randomized to therapy with ramipril (ACEI, n = 5) or ramipril plus losartan (CT, n = 6) or none (untreated, n = 8). Infarct size was similar between groups. At 8 weeks post-MI, myocytes were isolated from regions adjacent to and remote from the infarct to measure morphometric indices (cell volume, length, cross-sectional area, width) and parameters of contraction (% shortening and -dL/dt, rate of shortening) and relaxation (+dL/dt [rate of relengthening] and TR 70% [time for 70% relengthening]). Volume % collagen was measured from adjacent and remote regions. Adjacent myocyte volume was different between groups (2.5 +/- 0.1 x 10(4) microm(3) in CT, 3.0 +/- 0.4 x 10(4) microm(3) in ACEI, 3.5 +/- 0.2 x 10(4) microm(3) in untreated, analysis of variance [ANOVA] P =.001) as was length (158 +/- 4 microm, 161 +/- 9 microm, 189 +/- 8 microm, respectively, ANOVA P <.001). Adjacent cell volume and length in CT were lower than untreated (P <.05). Percent shortening and -dL/dt of isolated adjacent myocytes were improved with both ACEI (7.9 +/- 0.3%, -131 +/- 6 microm/sec, P <.05) and CT (7.7 +/- 0.3%, -144 +/- 8 microm/sec, P <.05) compared with no therapy (6.4 +/- 0.4%, -104 +/- 7 microm/sec), as was both +dL/dt and TR 70%. No between-group difference in volume % collagen was found in adjacent or remote regions. CONCLUSION: Compared with ACEI alone, the addition of ARB further limits adjacent noninfarcted myocyte hypertrophy during post-MI LV remodeling. Both ACEI alone and CT preserve isolated unloaded myocyte function, but neither significantly reduce interstitial collagen. The additional benefit of ARB on regional and global function in vivo may also be due to other factors including regional load.

Persistent stunning induces myocardial hibernation and protection: flow/function and metabolic mechanisms.

To test the hypothesis that persistent myocardial stunning can lead to hibernating myocardium, 13 pigs were chronically instrumented, and persistent stunning was induced regionally by 6 repetitive episodes of 90-minute coronary stenosis (CS) (30% reduction in baseline coronary blood flow [CBF]) followed by full reperfusion every 12 hours. During the 1st CS, CBF fell from 43+/-2 to 31+/-2 mL/min, and anterior wall thickening (AWT) fell by 54+/-8%, but posterior WT did not change. AWT never recovered fully and remained depressed by 31+/-7% before the 6th CS, reflecting persistent myocardial stunning, but baseline CBF was not changed. Surprisingly, during the 6th CS, AWT did not fall further despite a similar reduction in CBF during CS, as occurred with the 1st episode. Regional Mo2 fell similarly during the 1st and 6th CS. During the 1st CS, plasma glucose uptake increased, whereas free fatty acid (FFA) uptake was reduced. Before the 6th CS, glucose uptake remained elevated, whereas FFA uptake remained reduced. Histology revealed enhanced glycogen deposition, which could be explained by decreased glycogen synthase kinase (GSK)-3beta protein levels and activity. These results indicate that persistent stunning, even in the absence of chronic ischemia, can recapitulate the phenotype of myocardial hibernation. This results in a shift in the flow/function relationship where a 30% decrease in CBF is no longer accompanied by a fall in myocardial function, which could be explained, in part, by a shift in substrate utilization. These hemodynamic/metabolic adjustments could facilitate survival of hibernating myocardium.

Activation of Mst1 causes dilated cardiomyopathy by stimulating apoptosis without compensatory ventricular myocyte hypertrophy.

Activation of mammalian sterile 20-like kinase 1 (Mst1) by genotoxic compounds is known to stimulate apoptosis in some cell types. The importance of Mst1 in cell death caused by clinically relevant pathologic stimuli is unknown, however. In this study, we show that Mst1 is a prominent myelin basic protein kinase activated by proapoptotic stimuli in cardiac myocytes and that Mst1 causes cardiac myocyte apoptosis in vitro in a kinase activity-dependent manner. In vivo, cardiac-specific overexpression of Mst1 in transgenic mice results in activation of caspases, increased apoptosis, and dilated cardiomyopathy. Surprisingly, however, Mst1 prevents compensatory cardiac myocyte elongation or hypertrophy despite increased wall stress, thereby obscuring the use of the Frank-Starling mechanism, a fundamental mechanism by which the heart maintains cardiac output in response to increased mechanical load at the single myocyte level. Furthermore, Mst1 is activated by ischemia/reperfusion in the mouse heart in vivo. Suppression of endogenous Mst1 by cardiac-specific overexpression of dominant-negative Mst1 in transgenic mice prevents myocyte death by pathologic insults. These results show that Mst1 works as both an essential initiator of apoptosis and an inhibitor of hypertrophy in cardiac myocytes, resulting in a previously unrecognized form of cardiomyopathy.

Novel mechanisms mediating stunned myocardium.

Myocardial stunning is defined as the prolonged contractile dysfunction following an ischemic episode that does not result in necrosis, which also occurs in patients with coronary artery disease. There is also evidence to consider myocardial stunning as a fundamental component of hibernating myocardium. Various experimental approaches (from a brief episode to prolonged partial ischemia) and animal models (from rodents to large mammals) have been developed to investigate the pathogenesis of myocardial stunning. Three hypotheses to explain the mechanism, i.e. oxygen radical, Troponin I degradation, and Ca(2+), have been proposed. The first was tested primarily using large mammalian models, whereas the others were tested primarily using rodent models. Recently, the Ca(2+) handling hyothesis has been tested in a large mammalian swine model of myocardial stunning, in which both Ca(2+) and transients and L-type Ca(2+) current density were decreased. Relaxation function and phospholamban phosphorylation are also radically different in large mammalian and rodent models. In addition, troponin I degradation, which was identified as the mechanism of stunning in rodent models, was not found in stunned swine myocardium. Interestingly, the large mammalian model demonstrates that stunning elicits broad changes in gene and protein regulation, some of which have not been observed in the heart previously. The overall genomic adaptation upregulates the expression of survival genes that prevent irreversible damage. Pursuing these new concepts derived from large mammalian models of ischemia/reperfusion will provide more comprehensive mechanistic information underlying myocardial stunning and will serve to devise new therapeutic modalities for patients.