Early down-regulation of K+ channel genes and currents in the postinfarction heart

INTRODUCTION: Down-regulation of key K+ channel subunit gene expression and K+ currents is a universal response to cardiac hypertrophy, whatever the cause, including the postmyocardial infarction (post-MI) remodeled heart. METHODS AND RESULTS: We investigated the hypothesis that down-regulation of K+ channel genes and currents post-MI occurs early and before significant remodeled hypertrophy of the noninfarcted myocardium could be detected. We investigated (1) the incidence of induced ventricular tachyarrhythmias (VT) in 3-day post-MI rat heart; (2) action potential (AP) characteristics of isolated left ventricular (LV) myocytes from sham-operated and 3-day post-MI heart; (3) time course of changes in outward K+ currents Ito-fast(f) and I(K) in isolated myocytes from 3-day and 4-week post-MI noninfarcted LV and compared the changes with sham-operated animals; and (4) changes in the messenger and protein levels of Kv2.1, Kv4.2, and Kv4.3 in the LV and right ventricle of 3-day post-MI heart. Sustained VT was induced in 6 of 10 3-day post-MI rats and in none of 8 sham rats. The membrane capacitance of myocytes isolated from 3-day post-MI noninfarcted LV was not significantly different from control, whereas membrane capacitance 4-week post-MI was significantly higher, reflecting the development of hypertrophy. AP duration was increased and the density of Ito-f and I(K) were significantly decreased in 3-day post-MI LV myocytes compared with sham. The reduced density of Ito did not significantly differ in 4-week post-MI LV myocytes, whereas the density of I(K) was decreased further at 4 weeks post-MI. The changes in Ito-f and I(K) correlated with decreased messenger and protein levels of Kv4.2/Kv4.3 and Kv2.1, respectively. CONCLUSION: These results support the hypothesis that down-regulation of K+ channel gene expression and current in the post-MI LV occurs early and may be dissociated from the slower time course of post-MI remodeled hypertrophy. These changes may contribute to early arrhythmogenesis of the post-MI heart.     

Spatial alterations of Kv channels expression and K(+) currents in post-MI remodeled rat heart.

OBJECTIVE: The hypothesis being tested in the present study is that increased anisotropic properties occurs in the remodeled post-infarction heart due to spatial alterations in Kv channels expression and K(+) currents of the remodeled myocardium. METHODS: Three to 4 weeks post myocardial infarction (MI) in the rat, we measured the two components of the outward K(+) current, I(to-fast (f)) and I(to-slow(s)) in the epicardium (epi) and endocardium (endo) of noninfarcted remodeled left ventricle (LV) using patch clamp techniques. Alterations in mRNA and/or protein levels of potassium channel genes Kv1.4, Kv1.5, Kv2.1, Kv4.2 and Kv4.3 were measured in epi, midmyocardium (mid), and endo regions of LV and in the right ventricle (RV). RESULTS: In sham operated rat heart, the density of I(to-f) was 2.3 times greater in epi compared to endo myocytes. In post-MI heart, the density of I(to-f) and I(to-s) decreased to a similar degree in LV epi and endo but the difference in I(to-f) density between epi and endo persisted. The mRNA and/or protein levels of Kv1.4, Kv2.1, Kv4.2 and Kv4.3 but not Kv1.5 decreased to a varying extent in different regions of LV but not in RV of post-MI heart. CONCLUSIONS: Our results suggest that regional downregulation of Kv channels expression and density of K(+) currents can be a significant determinant of increased spatial electrophysiological heterogeneity and contribute to increased electrical instability of the post-MI heart.     

Alterations of sodium channel kinetics and gene expression in the postinfarction remodeled myocardium

INTRODUCTION: After a myocardial infarction (MI), the heart undergoes a remodeling process that includes hypertrophy of noninfarcted left ventricular myocytes. Alterations in the genetic expression, including reexpression of fetal isogene patterns, can result in electrophysiologic changes that contribute to the arrhythmogenicity of post-MI heart. The present study investigated possible alterations in gene expression of Na+ channel subtypes, as well as the kinetics of the Na+ current (I(Na)), in 3- to 4-week-old post-MI rat remodeled left ventricular myocardium. METHODS AND RESULTS: Using a macropatch technique, we showed increased Na+ channel bursting activity during sustained depolarization in post-MI remodeled myocytes resulting in a large slow component of the I(Na) decay. A tetrodotoxin-sensitive current contributed 18% to the prolonged APD90 of isolated post-MI myocytes compared with 6% in control myocytes. Our molecular studies revealed that, in addition to the rat heart I (rH I) subtype, thought to be the predominant subtype that encodes a tetrodotoxin-resistant isoform, the brain subtypes NaCh I and NaCh Ia also are expressed in the rat myocytes. Post-MI remodeled myocardium showed increased expression of NaCh I protein with reversion of the NaCh Ia/NaCh I isoform ratio toward the fetal phenotype. CONCLUSION: Our findings raise the possibility that the increase in the slow component of I(Na) in post-MI remodeled myocytes is secondary to the increased expression of NaCh I. Additional studies are required to address these questions and to characterize the functional role of the NaCh I subtypes in cardiac myocytes.     

Alterations in myofilament function contribute to left ventricular dysfunction in pigs early after myocardial infarction

Myocardial infarction (MI) initiates cardiac remodeling, depresses pump function, and predisposes to heart failure. This study was designed to identify early alterations in Ca2+ handling and myofilament proteins, which may contribute to contractile dysfunction and reduced beta-adrenergic responsiveness in postinfarct remodeled myocardium. Protein composition and contractile function of skinned cardiomyocytes were studied in remote, noninfarcted left ventricular (LV) subendocardium from pigs 3 weeks after MI caused by permanent left circumflex artery (LCx) ligation and in sham-operated pigs. LCx ligation induced a 19% increase in LV weight, a 69% increase in LV end-diastolic area, and a decrease in ejection fraction from 54+/-5% to 35+/-4% (all P<0.05), whereas cardiac responsiveness to exercise-induced increases in circulating noradrenaline levels was blunted. Endogenous protein kinase A (PKA) was significantly reduced in remote myocardium of MI animals, and a negative correlation (R=0.62; P<0.05) was found between cAMP levels and LV weight-to-body weight ratio. Furthermore, SERCA2a expression was 23% lower after MI compared with sham. Maximal isometric force generated by isolated skinned myocytes was significantly lower after MI than in sham (15.4+/-1.5 versus 19.2+/-0.9 kN/m2; P<0.05), which might be attributable to a small degree of troponin I (TnI) degradation observed in remodeled postinfarct myocardium. An increase in Ca2+ sensitivity of force (pCa50) was observed after MI compared with sham (DeltapCa50=0.17), which was abolished by incubating myocytes with exogenous PKA, indicating that the increased Ca2+ sensitivity resulted from reduced TnI phosphorylation. In conclusion, remodeling of noninfarcted pig myocardium is associated with decreased SERCA2a and myofilament function, which may contribute to depressed LV function. The full text of this article is available online at http://circres.ahajournals.org.     

心肌梗死后钾通道Kv2.1基因表达的变化

目的研究大鼠心肌梗死(MI)后钾通道Kv2.1的动态变化。方法通过结扎大鼠左前降支近端建立MI模型,手术后存活大鼠进入MI组(存活大鼠又等分为两个亚组,分别于7天和30天时取心脏标本,依取标本的时间归入7天组和30天组)。同时,设立相应的假手术(SH)组。应用半定量逆转录-聚合酶链反应方法和Western Blots方法检测左室心肌(MI者取非MI区左室心肌)钾通道Kv2.1mRNA及通道蛋白量。结果与SH组比较,MI组(7天组、30天组)Kv2.1mRNA和蛋白量明显下降(P<0.05或P<0.01)。MI组内30天组与7天组比较,Kv2.1mRNA和蛋白量显著下降(P<0.05)。结论MI后钾通道Kv2.1基因表达呈时间依赖性下调,此可能是MI后易发心律失常的分子机制之一。     

Effects of the calcineurin dependent signaling pathway inhibition by cyclosporin A on early and late cardiac remodeling following myocardial infarction

BACKGROUND: The calcineurin-mediated signaling pathway has been implicated as one of the crucial pathways in cardiac hypertrophy. However, the role of calcineurin pathway on cardiac remodeling after myocardial infarction (MI) has not been well defined. METHODS: Infarcted rats (n = 45) were randomized into calcineurin inhibitor, cyclosporin A (CsA) or vehicle groups, 3 days after MI and treated for 2 weeks (early post-MI cardiac remodeling stage), or randomized 17 days after MI and treated for 2 weeks (late remodeling stage). RESULTS: Calcineurin pathway inhibition during the early cardiac remodeling stage attenuated the myocardial hypertrophy after MI (P < 0.05). However, left ventricular dimensions were further increased and fractional shortening deteriorated with calcineurin inhibition during this stage (P < 0.05, each). During late remodeling stage, CsA treatment did not affect myocardial hypertrophy and cardiac dilation following MI. CONCLUSION: Our results strongly support the hypothesis that calcineurin pathway mediates compensatory myocardial hypertrophy during the early remodeling stage after MI. However, the calcineurin pathway does not seem to affect the late remodeling after MI.     

Reexpression of T-type Ca2+ channel gene and current in post-infarction remodeled rat left ventricle

OBJECTIVE: T-type Ca2+ currents (I(Ca-T)) are present in neonatal rat myocytes but is not detected in adult ventricular myocytes. The present study was designed to investigate the expression of the T-type Ca2+ channel gene and current in post-infarction remodeled hypertrophied rat left ventricle (LV). METHODS: We compared the expression of T-type Ca2+ channel gene alpha-1G in neonatal rat LV, in adult sham-operated LV and remodeled hypertrophied LV 3 to 4 weeks post-myocardial infarction (MI) using RNase protection assay (RPA). The cDNA fragment of alpha-1G used in RPA was obtained from poorly conserved region of recently published T-type Ca2+ channel coding sequence of rat by RT-PCR. The fragment was verified by restriction enzyme digestion and sequencing. The presence of I(Ca-T) in LV of sham and post-MI rats was examined using patch-clamp techniques. In the presence of K+-free, Na+-free external solution, I(Ca-T) was separated from I(Ca-L) by different holding potentials (HP). I(Ca-T) was also recorded during depolarization to -40 mV from a HP of -80 mV with NaCl in external solution and I(Na) suppressed by 100 microM tetrodotoxin (TTX). RESULTS: The T-type Ca2+ channel gene alpha-1G was expressed in neonatal heart, the expression level decreased by 80%, in adult sham heart and was reexpressed in MI (158% increases compared to sham; P<0.01). I(Ca-T) was recorded in 11 of 31 MI cells in presence of K+-free, Na+-free external solution and in 9 of 14 cells when I(Na) was suppressed by TTX. I(Ca-T) was not detected in any of 21 sham cells. I(Ca-T) density was 1.1+/-0.4 pA/pF. I(Ca-T) was more sensitive to Ni2+ and less sensitive to nisoldipine. CONCLUSIONS: T-type Ca2+ channel gene and current are reexpressed in rat post-MI remodeled LV myocytes. Its functional significance in the post-MI remodeling process remains to be defined.     

Treatment with dimethylthiourea prevents left ventricular remodeling and failure after experimental myocardial infarction in mice: role of oxidative stress

Oxidative stress might play an important role in the progression of left ventricular (LV) remodeling and failure that occur after myocardial infarction (MI). We determined whether reactive oxygen species (ROS) are increased in the LV remodeling and failure in experimental MI with the use of electron spin resonance spectroscopy and whether the long-term administration of dimethylthiourea (DMTU), hydroxyl radical (.OH) scavenger, could attenuate these changes. We studied 3 groups of mice: sham-operated (sham), MI, and MI animals that received DMTU (MI+DMTU). Drugs were administered to the animals daily via intraperitoneal injection for 4 weeks.OH was increased in the noninfarcted myocardium from MI animals, which was abolished in MI+DMTU. Fractional shortening was depressed by 65%, LV chamber diameter was increased by 53%, and the thickness of noninfarcted myocardium was increased by 37% in MI. MI+DMTU animals had significantly better LV contractile function and smaller increases in LV chamber size and hypertrophy than MI animals. Changes in myocyte cross-sectional area determined with LV mid-free wall specimens were concordant with the wall thickness data. Collagen volume fraction of the noninfarcted myocardium showed significant increases in the MI, which were also attenuated with DMTU. Myocardial matrix metalloproteinase-2 activity, measured with gelatin zymography, was increased with MI after 7 and 28 days, which was attenuated in MI+DMTU. Thus, the attenuation of increased myocardial ROS and metalloproteinase activity with DMTU may contribute, at least in part, to its beneficial effects on LV remodeling and failure. Therapies designed to interfere with oxidative stress might be beneficial to prevent myocardial failure.     

MCIP1 overexpression suppresses left ventricular remodeling and sustains cardiac function after myocardial infarction

Pathological remodeling of the left ventricle (LV) after myocardial infarction (MI) is a major cause of heart failure. Although cardiac hypertrophy after increased loading conditions has been recognized as a clinical risk factor for human heart failure, it is unknown whether post-MI hypertrophic remodeling of the myocardium is beneficial for cardiac function over time, nor which regulatory pathways play a crucial role in this process. To address these questions, transgenic (TG) mice engineered to overexpress modulatory calcineurin-interacting protein-1 (MCIP1) in the myocardium were used to achieve cardiac-specific inhibition of calcineurin activation. MCIP1-TG mice and their wild-type (WT) littermates, were subjected to MI and analyzed 4 weeks later. At 4 weeks after MI, calcineurin was activated in the LV of WT mice, which was significantly reduced in MCIP1-TG mice. WT mice displayed a 78% increase in LV mass after MI, which was reduced by 38% in MCIP1-TG mice. Echocardiography indicated marked LV dilation and loss of systolic function in WT-MI mice, whereas TG-MI mice displayed a remarkable preservation of LV geometry and contractility, a pronounced reduction in myofiber hypertrophy, collagen deposition, and beta-MHC expression compared with WT-MI mice. Together, these results reveal a protective role for MCIP1 in the post-MI heart and suggest that calcineurin is a crucial regulator of postinfarction-induced pathological LV remodeling. The improvement in functional, structural, and molecular abnormalities in MCIP1-TG mice challenges the adaptive value of post-MI hypertrophy of the remote myocardium. The full text of this article is available online at http://circres.ahajournals.org.     

Myocyte contractile function is intact in the post-infarct remodeled rat heart despite molecular alterations

OBJECTIVE: To investigate the cellular mechanisms underlying global and regional LV dysfunction in the post-infarct (MI) remodeled rat hearts. METHODS: LV remodeling and function were quantified by echocardiography, morphometry, in vivo hemodynamics, and isolated perfused heart studies in 6 weeks post-MI and sham-operated rats. LV myocytes from sham and MI hearts were used for morphometric and functional studies. Myocyte contractile function and intracellular calcium kinetics were measured at different stimulation frequencies (0.2-2 Hz), temperatures (30 and 37 degrees C), and external viscous load (1, 15, 200 and 300 centipoise). Myocyte apoptosis was measured by DNA laddering; BCL-2, BAX, Na(+)-Ca(2+) exchanger, and SERCA-2 proteins by western blot; and brain natriuretic peptide (BNP), SERCA-2 mRNA by RT-PCR. RESULTS: MI hearts were remodeled (Echo LV diameter 7.3+/-0.38 vs. 5.9+/-0.16 mm, P<0.03), and showed global (Echo % fractional shortening 30+/-2.4 vs. 58+/-3, P<0.001), and regional contractile dysfunction of non-infarcted myocardium (Echo % systolic posterior wall thickening 36+/-2 vs. 57+/-1.7, P<0.001). In vivo hemodynamic and isolated heart function studies confirmed depressed LV systolic and diastolic function and increased volumes. Whereas, myocytes isolated from infarcted hearts were remodeled (40% longer and 10% wider), their contractile function and calcium kinetics under basal conditions and at high stimulation frequency, temperature and viscous load were similar to sham myocytes. The mRNA for BNP was increased whereas that for SERCA-2 decreased, but the SERCA-2 protein was normal. Despite myocyte hypertrophy, ventricular septal thickness was reduced in infarcted hearts (2.2+/-0.1 vs. 2. 6+/-0.07 mm, P<0.01), and showed increased apoptosis. CONCLUSIONS: Myocytes from remote non-infarcted myocardium of the remodeled hearts have normal contractile function, despite structural remodeling and altered gene expression. Non-myocyte factors may be more important in genesis of contractile dysfunction in the remodeled heart, for up to 6 weeks after MI.     

Inhibition of TGF-beta signaling exacerbates early cardiac dysfunction but prevents late remodeling after infarction

OBJECTIVE: Transforming growth factor (TGF)-beta promotes the deposition of extracellular matrix protein and also acts as an anti-inflammatory cytokine. These biological effects might be involved in the development and progression of left ventricular (LV) remodeling and failure after myocardial infarction (MI). However, its pathophysiological significance remains obscure in post-MI hearts. METHODS: Anterior MI was produced in mice by ligating the left coronary artery. TGF-beta mRNA levels increased in both infarcted and noninfarcted LV after MI. To block TGF-beta signaling during the early phase of MI, an extracellular domain of TGF-beta type II receptor (TbetaIIR) plasmid was transfected into the limb skeletal muscles 7 days before ligation. RESULTS: TbetaIIR increased the mortality during 24 h of MI, as well as exacerbated LV dilatation and contractile dysfunction, the infiltration of neutrophils, and gene expression of tumor necrosis factor-alpha, interleukin-1beta, and monocyte chemoattractant protein-1 compared with nontreated MI mice despite the comparable infarct size. Next, to block TGF-beta signaling during the later phase, TbetaIIR was transfected into mice at days 0 and 7 after ligation. At 4 weeks, LV dilatation and contractile dysfunction in association with myocyte hypertrophy and interstitial fibrosis of noninfarcted LV seen in MI mice were prevented by TbetaIIR. CONCLUSIONS: The activation of TGF-beta is protective against ischemic myocardial damage during the early phase. However, the beneficial effects might be lost, when its expression is sustained, thereby leading to LV remodeling and failure after MI.     

Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A.

The immunosuppressive agents cyclosporin A (CsA) and FK 506 bind to distinct families of intracellular proteins (immunophilins) termed cyclophilins and FK 506-binding proteins (FKBPs). Recently, it has been shown that, in vitro, the complexes of CsA-cyclophilin and FK 506-FKBP-12 bind to and inhibit the activity of calcineurin, a calcium-dependent serine/threonine phosphatase. We have investigated the effects of drug treatment on phosphatase activity in T lymphocytes. Calcineurin is expressed in T cells, and its activity can be measured in cell lysates. Both CsA and FK 506 specifically inhibit cellular calcineurin at drug concentrations that inhibit interleukin 2 production in activated T cells. Rapamycin, which binds to FKBPs but exhibits different biological activities than FK 506, has no effect on calcineurin activity. Furthermore, excess concentrations of rapamycin prevent the effects of FK 506, apparently by displacing FK 506 from FKBPs. These results show that calcineurin is a target of drug-immunophilin complexes in vivo and establish a physiological role for calcineurin in T-cell activation.     

Infarct Size Reduction and Attenuation of Global Left Ventricular Remodeling with the CorCap<Superscript>TM</Superscript> Cardiac Support Device Following Acute Myocardial Infarction in Sheep

Background:Whether mechanical restraint of the left ventricle (LV) can influence remodeling following myocardial infarction (MI) remains poorly understood. The following discussion details three studies examining the effects of surgically placing a cardiac support device (CSD) over the entire epicardial surface, on infarct expansion, global cardiac function and myocyte geometry and function post-MI. Methods: The effects of passive constraint on infarct expansion and global cardiac function/myocardial energetics were investigated in 10 sheep (5 MI only; 5 MI + CSD) using pressure-volume analysis and magnetic resonance imaging (MRI). Additionally, 11 sheep (5 MI only; 6 MI + CSD) were used to study the effects of passive restraint on myocyte geometry and function post-MI, with 10 additional uninstrumented sheep serving as controls. Baseline data was collected followed by the creation of an anterior infarct. 1 week post-infarct the animals underwent a second set of data collection studies followed by placement of the CSD in the experimental groups. Additional data was collected at 2 and 3 months post-MI. The animals in the myocyte function group underwent additional studies immediately following the 3 month time point. Results: Infarct expansion was diminished as a result of the CSD. At 1 week post-MI the akinetic area was similar in both groups. At the terminal time-point, the akinetic area in the control group was similar to the 1-week time-point whereas, in the CSD group, the area of akinesis decreased (P = 0.001). A comparison of the two groups at the terminal time-point demonstrates a significantly diminished area of akinesis in the CSD group (P = 0.004). The relative area of akinesis followed a similar pattern. The CSD group also exhibited a decrease in end-diastolic volume (control 110.3 ± 19.8 mL vs. CSD 67.6 ± 4.7 mL, P = .006) and an improved ejection fraction (control 15.5% ± 5.7% vs. CSD 29.46% ± 4.42%, P = .008) relative to the control group. Myocardial energetics were also enhanced in the CSD group as evidenced by significant improvements in potential energy (control 2015 ± 503 mL ⋅ mm Hg/beat vs. CSD 885 ± 220 mL ⋅ Hg/beat, P = .006), efficiency (control 39.4% ± 13.6% vs. CSD 59.8% ± 8.5%, P = .044), and oxygen consumption (control 0.072 ± 0.013 mL O₂/beat vs. CSD 0.052 ± 0.007 mL O₂/beat, P = .034). Isolated LV myocyte shortening velocity was reduced by 35% from control values (P < 0.05) in both MI groups. LV myocyte β-adrenergic response was reduced with MI, but normalized in the MI + CSD group. Relative collagen content was increased and matrix metalloproteinase-9 was decreased within the MI border region of the CSD group. Conclusions:The CorCap cardiac support device retarded infarct expansion, improved global and regional cardiac function and beneficially modified LV and myocyte remodeling post-MI. These findings provide evidence that non-pharmacological strategies can interrupt adverse LV remodeling post-MI.     

Streptozotocin-induced hyperglycemia exacerbates left ventricular remodeling and failure after experimental myocardial infarction

OBJECTIVES: The aim of the present study was to determine whether streptozotocin (STZ)-induced hyperglycemia exacerbates progressive left ventricular (LV) dilation and dysfunction after myocardial infarction (MI). BACKGROUND: Diabetes mellitus (DM) adversely affects the outcomes in patients with MI. However, it is unknown whether DM can directly affect the development of post-MI LV remodeling and failure. METHODS: Male mice were injected intraperitoneally with STZ (200 mg/kg; DM group) or vehicle only. At two weeks, MI was created in the STZ-injected (DM+MI group) or vehicle-injected mice (MI group) by left coronary artery ligation, and they were followed up for another four weeks. RESULTS: Survival during six weeks was significantly lower in the DM+MI versus MI group (25% vs. 71%; p < 0.01), despite a similar infarct size (60 +/- 2% vs. 61 +/- 2%; p = NS). Echocardiography after two weeks of ligation showed LV dilation and dysfunction with MI, both of which were exaggerated in the DM+MI group. Likewise, LV end-diastolic pressure and lung weight were increased in mice with MI, and this increase was enhanced in the DM+MI group. The myocyte cross-sectional area in the non-infarcted LV increased to a similar degree in the DM+MI and MI groups, whereas the collagen volume fraction was greater in the DM+MI group. Deoxyribonucleic acid laddering was greater in the DM+MI group. CONCLUSIONS: Hyperglycemia decreased survival and exaggerated LV remodeling and failure after MI by increasing interstitial fibrosis and myocyte apoptosis. Diabetes mellitus could be a risk factor for heart failure, independent of coronary artery lesions.     

Effects of cyclosporin A on water-immersion stress-induced gastric lesion and gastric secretion in rats

Cyclosporin A is an immunosuppressive agent which is well known as a specific inhibitor of calcineurin (protein phosphatase 2B). In this study, we investigated the effects of cyclosporin A on water-immersion stress-induced gastric ulcer formation and gastric acid secretion in rats. We also examined the localization of calcineurin immunohistochemically. Calcineurin was specifically expressed in gastric parietal cells and chief cells of the gastric mucosa. The intraperitoneal administration of cyclosporin A dose-dependently suppressed the development of gastric mucosal lesions induced by water-immersion stress and inhibited gastric acid secretion, as assessed by pylorus ligation. These results indicated that calcineurin may play an important role in gastric acid secretion. Received: October 14, 1999 / Accepted: January 28, 2000     

Regulatory interactions of calmodulin-binding proteins: phosphorylation of calcineurin by autophosphorylated Ca2+/calmodulin-dependent protein kinase II.

The Ca2+/calmodulin (CaM)-dependent protein phosphatase calcineurin is rapidly phosphorylated (0.8 mol of 32PO4 per mol of 60-kDa subunit of calcineurin) by brain Ca2+/CaM-dependent protein kinase II (CaM-kinase II). This reaction requires the autophosphorylated, Ca2+-independent form of CaM-kinase II since Ca2+/CaM binding to calcineurin inhibits phosphorylation. However, the phosphorylation reaction does require Ca2+, presumably acting through the 19-kDa subunit of calcineurin. Calcineurin is a good substrate for CaM-kinase II, with a Km of 19 microM and Vmax of 2.4 mumol/min per mg. Phosphorylation of calcineurin changed its phosphatase activity with either a 2-fold increase in Km (32P-labeled myosin light chain as substrate) or a 50% decrease in Vmax (p-nitrophenyl phosphate as substrate). The phosphorylated calcineurin exhibited very slow autodephosphorylation (0.09 nmol/min per mg) but was effectively dephosphorylated by brain protein phosphatase IIA. Dephosphorylation, like phosphorylation, was blocked by high concentrations of Ca2+/CaM and stimulated by Ca2+ alone. Thus calcineurin has a regulatory phosphorylation site that is phosphorylated by the Ca2+-independent form of CaM-kinase II and blocked by high concentrations of Ca2+/CaM.     

Interleukin-10 improves left ventricular function in rats with heart failure subsequent to myocardial infarction

Evidence has shown that pro-inflammatory cytokines, especially TNF-alpha, are involved in the inflammatory response in the remodelling process after myocardial infarction (MI). Although IL-10, an anti-inflammatory cytokine, has been shown to antagonize some of the deleterious effects of TNF-alpha, little is known about its role in post-MI left ventricular (LV) dysfunction. The aim of the present study was to investigate whether a therapy with rhIL-10 could be beneficial in an animal model of post-MI heart failure (HF). Rats with experimental MI were treated with rhIL-10 (75 microg/kg/d sc) starting directly after MI induction, and continuing for 4 weeks. Controls were untreated MI and sham-operated rats. Cardiac function was assessed by echocardiography and cardiac catheterization 4 weeks after MI induction. Membrane-bound and soluble fractions of TNF-alpha, IL-6 and IL-10, the ratio of TNF-alpha to IL-10, serum levels of MCP-1 as well as myocardial macrophage infiltration, were analyzed. Treatment with rhIL-10 significantly improved post-MI LV function (FS +127%;, dP/dt(max) +131%; LVEDP -36%). This effect was associated with a significant decrease in pro-inflammatory cytokine and chemokine levels (TNF-alpha, IL-6, MCP-1) and furthermore resulted in a reduced myocardial infiltration of macrophages.