Chronic CaMKII inhibition blunts the cardiac contractile response to exercise training

Activation of the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a critical role modulating cardiac function in both health and disease. Here, we determined the effect of chronic CaMKII inhibition during an exercise training program in healthy mice. CaMKII was inhibited by KN-93 injections. Mice were randomized to the following groups: sham sedentary, sham exercise, KN-93 sedentary, and KN-93 exercise. Cardiorespiratory function was evaluated by ergospirometry during treadmill running, echocardiography, and cardiomyocyte fractional shortening and calcium handling. The results revealed that KN-93 alone had no effect on exercise capacity or fractional shortening. In sham animals, exercise training increased maximal oxygen uptake by 8% (p < 0.05) compared to a 22% (p < 0.05) increase after exercise in KN-93 treated mice (group difference p < 0.01). In contrast, in vivo fractional shortening evaluated by echocardiography improved after exercise in sham animals only: from 25 to 32% (p < 0.02). In inactive mice, KN-93 reduced rates of diastolic cardiomyocyte re-lengthening (by 25%, p < 0.05) as well as Ca2+ transient decay (by 16%, p < 0.05), whereas no such effect was observed after exercise training. KN-93 blunted exercise training response on cardiomyocyte fractional shortening (63% sham vs. 18% KN-93; p < 0.01 and p < 0.05, respectively). These effects could not be solely explained by the Ca2+ transient amplitude, as KN-93 reduced it by 20% (p < 0.05) and response to exercise training was equal (64% sham and 47% KN-93; both p < 0.01). We concluded that chronic CaMKII inhibition increased time to 50% re-lengthening which were recovered by exercise training, but paradoxically led to a greater increase in maximal oxygen uptake compared to sham mice. Thus, the effect of chronic CaMKII inhibition is multifaceted and of a complex nature.

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title: Role of CaMKII in RyR leak, EC coupling and action potential duration: a computational model.
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title: Calcium, calmodulin, and calcium-calmodulin kinase II: heartbeat to heartbeat and beyond.
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title: CaMKII and Its Role in Cardiac Arrhythmia
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title: Calcium/Calmodulin-Dependent Protein Kinase II Contributes to Cardiac Arrhythmogenesis in Heart Failure
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title: Calmodulin kinase signaling in heart: an intriguing candidate target for therapy of myocardial dysfunction and arrhythmias.
abstract: The multifunctional Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) has emerged as a proarrhythmic and procardiomyopathic signal in a wide range of structural heart diseases. This review discusses CaMKII structure and function and recent evidence implicating CaMKII inhibition as a potential strategy for treating myocardial dysfunction and arrhythmias in the setting of structural heart disease.

title: KN-93, an inhibitor of multifunctional Ca++/calmodulin-dependent protein kinase, decreases early afterdepolarizations in rabbit heart.
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title: Death, Cardiac Dysfunction, and Arrhythmias Are Increased by Calmodulin Kinase II in Calcineurin Cardiomyopathy
abstract: Activation of cellular Ca2+ signaling molecules appears to be a fundamental step in the progression of cardiomyopathy and arrhythmias. Myocardial overexpression of the constitutively active Ca2+-dependent phosphatase calcineurin (CAN) causes severe cardiomyopathy marked by left ventricular (LV) dysfunction, arrhythmias, and increased mortality rate, but CAN antagonist drugs primarily reduce hypertrophy without improving LV function or risk of death.  We found that activity and expression of a second Ca2+-activated signaling molecule, calmodulin kinase II (CaMKII), were increased in hearts from CAN transgenic mice and that CaMKII-inhibitory drugs improved LV function and suppressed arrhythmias. We devised a genetic approach to “clamp” CaMKII activity in CAN mice to control levels by interbreeding CAN transgenic mice with mice expressing a specific CaMKII inhibitor in cardiomyocytes. We developed transgenic control mice by interbreeding CAN transgenic mice with mice expressing an inactive version of the CaMKII-inhibitory peptide. CAN mice with CaMKII inhibition had reduced risk of death and increased LV and ventricular myocyte function and were less susceptible to arrhythmias. CaMKII inhibition did not reduce transgenic overexpression of CAN or expression of endogenous CaMKII protein or significantly reduce most measures of cardiac hypertrophy.  CaMKII is a downstream signal in CAN cardiomyopathy, and increased CaMKII activity contributes to cardiac dysfunction, arrhythmia susceptibility, and longevity during CAN overexpression.

title: Calmodulin kinase II inhibition protects against structural heart disease
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title: Requirement for Ca2+/calmodulin-dependent kinase II in the transition from pressure overload-induced cardiac hypertrophy to heart failure in mice.
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title: Ryanodine Receptor Phosphorylation by Calcium/Calmodulin-Dependent Protein Kinase II Promotes Life-Threatening Ventricular Arrhythmias in Mice With Heart Failure
abstract: Approximately half of patients with heart failure die suddenly as a result of ventricular arrhythmias. Although abnormal Ca2+ release from the sarcoplasmic reticulum through ryanodine receptors (RyR2) has been linked to arrhythmogenesis, the molecular mechanisms triggering release of arrhythmogenic Ca2+ remain unknown. We tested the hypothesis that increased RyR2 phosphorylation by Ca2+/calmodulin-dependent protein kinase II is both necessary and sufficient to promote lethal ventricular arrhythmias.  Mice in which the S2814 Ca2+/calmodulin-dependent protein kinase II site on RyR2 is constitutively activated (S2814D) develop pathological sarcoplasmic reticulum Ca2+ release events, resulting in reduced sarcoplasmic reticulum Ca2+ load on confocal microscopy. These Ca2+ release events are associated with increased RyR2 open probability in lipid bilayer preparations. At baseline, young S2814D mice have structurally and functionally normal hearts without arrhythmias; however, they develop sustained ventricular tachycardia and sudden cardiac death on catecholaminergic provocation by caffeine/epinephrine or programmed electric stimulation. Young S2814D mice have a significant predisposition to sudden arrhythmogenic death after transverse aortic constriction surgery. Finally, genetic ablation of the Ca2+/calmodulin-dependent protein kinase II site on RyR2 (S2814A) protects mutant mice from pacing-induced arrhythmias versus wild-type mice after transverse aortic constriction surgery.  Our results suggest that Ca2+/calmodulin-dependent protein kinase II phosphorylation of RyR2 Ca2+ release channels at S2814 plays an important role in arrhythmogenesis and sudden cardiac death in mice with heart failure.

title: Interval Training Normalizes Cardiomyocyte Function, Diastolic Ca2+ Control, and SR Ca2+ Release Synchronicity in a Mouse Model of Diabetic Cardiomyopathy
abstract: In the present study we explored the mechanisms behind excitation–contraction (EC) coupling defects in cardiomyocytes from mice with type-2 diabetes (db/db).  We determined whether 13 weeks of aerobic interval training could restore cardiomyocyte Ca2+ cycling and EC coupling.  Reduced contractility in cardiomyocytes isolated from sedentary db/db was associated with increased diastolic sarcoplasmic reticulum (SR)-Ca2+ leak, reduced synchrony of Ca2+ release, reduced transverse (T)-tubule density, and lower peak systolic and diastolic Ca2+ and caffeine-induced Ca2+ release. Additionally, the rate of SR Ca2+ ATPase–mediated Ca2+ uptake during diastole was reduced, whereas a faster recovery from caffeine-induced Ca2+ release indicated increased Na+/Ca2+-exchanger activity. The increased SR-Ca2+ leak was attributed to increased Ca2+-calmodulin–dependent protein kinase (CaMKII&dgr;) phosphorylation, supported by the normalization of SR-Ca2+ leak on inhibition of CaMKII&dgr; (AIP). Exercise training restored contractile function associated with restored SR Ca2+ release synchronicity, T-tubule density, twitch Ca2+ amplitude, SR Ca2+ ATPase and Na+/Ca2+-exchanger activities, and SR-Ca2+ leak. The latter was associated with reduced phosphorylation of cytosolic CaMKII&dgr;. Despite normal contractile function and Ca2+ handling after the training period, phospholamban was hyperphosphorylated at Serine-16. Protein kinase A inhibition (H-89) in cardiomyocytes from the exercised db/db group abolished the differences in SR-Ca2+ load when compared with the sedentary db/db mice. EC coupling changes were observed without changes in serum insulin or glucose levels, suggesting that the exercise training–induced effects are not via normalization of the diabetic condition.  These data demonstrate that aerobic interval training almost completely restored the contractile function of the diabetic cardiomyocyte to levels close to sedentary wild type.

title: CaMKII Negatively Regulates Calcineurin–NFAT Signaling in Cardiac Myocytes
abstract: Pathological cardiac myocyte hypertrophy is thought to be induced by the persistent increases in intracellular Ca2+ needed to maintain cardiac function when systolic wall stress is increased. Hypertrophic Ca2+ binds to calmodulin (CaM) and activates the phosphatase calcineurin (Cn) and CaM kinase (CaMK)II. Cn dephosphorylates cytoplasmic NFAT (nuclear factor of activated T cells), inducing its translocation to the nucleus where it activates antiapoptotic and hypertrophic target genes. Cytoplasmic CaMKII regulates Ca2+ handling proteins but whether or not it is directly involved in hypertrophic and survival signaling is not known.  This study explored the hypothesis that cytoplasmic CaMKII reduces NFAT nuclear translocation by inhibiting the phosphatase activity of Cn.  Green fluorescent protein–tagged NFATc3 was used to determine the cellular location of NFAT in cultured neonatal rat ventricular myocytes (NRVMs) and adult feline ventricular myocytes. Constitutively active (CaMKII-CA) or dominant negative (CaMKII-DN) mutants of cytoplasmic targeted CaMKII&dgr;c were used to activate and inhibit cytoplasmic CaMKII activity. In NRVM CaMKII-DN (48.5±3%, P<0.01 versus control) increased, whereas CaMKII-CA decreased (5.9±1%, P<0.01 versus control) NFAT nuclear translocation (Control: 12.3±1%). Cn inhibitors were used to show that these effects were caused by modulation of Cn activity. Increasing Ca2+ increased Cn-dependent NFAT translocation (to 71.7±7%, P<0.01) and CaMKII-CA reduced this effect (to 17.6±4%). CaMKII-CA increased TUNEL and caspase-3 activity (P<0.05). CaMKII directly phosphorylated Cn at Ser197 in CaMKII-CA infected NRVMs and in hypertrophied feline hearts.  These data show that activation of cytoplasmic CaMKII inhibits NFAT nuclear translocation by phosphorylation and subsequent inhibition of Cn.

title: Constitutively active calcineurin in skeletal muscle increases endurance performance and mitochondrial respiratory capacity.
abstract: Expression of an activated form of calcineurin in skeletal muscle selectively up-regulates slow-fiber-specific gene expression. Here, we tested the hypothesis that expression of activated calcineurin in skeletal muscle influences body composition, energy homeostasis, and exercise performance. Using transgenic mice expressing activated calcineurin (CnA*) in skeletal muscle (MCK-CnA* transgenic mice), we determined whether skeletal muscle reprogramming by calcineurin activation affects exercise performance and skeletal muscle mitochondrial function. Body weight and extensor digitorum longus (EDL) skeletal muscle weight were reduced 10% in MCK-CnA* mice compared with wild-type littermates. Basal oxygen consumption, food intake, and voluntary exercise behavior were unchanged between MCK-CnA* and wild-type mice. However, when total energy expenditure was normalized by fat-free mass, energy expenditure was increased in MCK-CnA* mice. An endurance performance treadmill running test revealed MCK-CnA* mice are fatigue resistant and run 50% farther before exhaustion. After a standardized exercise bout, glycogen and triglyceride content in EDL muscle was higher in MCK-CnA* vs. wild-type mice. Mitochondrial respiratory capacity was increased 35% in EDL muscle from resting MCK-CnA* mice. In conclusion, our results provide evidence to support the hypothesis that calcineurin activation in skeletal muscle increases mitochondrial oxidative function and energy substrate storage, which contributes to enhanced endurance exercise performance. These adaptive changes occur as a consequence of a lifelong expression of a constitutively active calcineurin and mimic the response to chronic endurance training.

title: miR-499 regulates mitochondrial dynamics by targeting calcineurin and dynamin-related protein-1
abstract: Myocardial infarction is a leading cause of mortality worldwide. Here we report that modulation of microRNA-499 (miR-499) levels affects apoptosis and the severity of myocardial infarction and cardiac dysfunction induced by ischemia-reperfusion. We found that both the α- and β-isoforms of the calcineurin catalytic subunit are direct targets of miR-499 and that miR-499 inhibits cardiomyocyte apoptosis through its suppression of calcineurin-mediated dephosphorylation of dynamin-related protein-1 (Drp1), thereby decreasing Drp1 accumulation in mitochondria and Drp1-mediated activation of the mitochondrial fission program. We also found that p53 transcriptionally downregulates miR-499 expression. Our data reveal a role for miR-499 in regulating the mitochondrial fission machinery and we suggest that modulation of miR-499 levels may provide a therapeutic approach for treating myocardial infarction.

title: Aerobic exercise reduces cardiomyocyte hypertrophy and increases contractility, Ca2+ sensitivity and SERCA-2 in rat after myocardial infarction.
abstract: Although it is generally accepted that endurance training improves cardiac function after myocardial infarction the sub-cellular mechanisms are uncertain. The present study reports the effects of aerobic endurance training on myocardial mass, myocyte dimensions, contractile function, Ca2+ handling, and myofilament responsiveness to Ca2+ in cardiomyocytes from healthy and failing rat hearts.



Adult female Sprague-Dawley rats ran on a treadmill 1.5 h/day, 5 days a week for 8 weeks. Exercise intervals alternated between 8 min at 85-90% of V(O(2max)) and 2 min at 50-60%. Training started 4 weeks after ligation of the left coronary artery (TR-INF, n=11) or sham operation (TR-SHAM, n=6). Sedentary animals (SED-SHAM, n=6; SED-INF, n=13) were controls.



After 6 weeks V(O(2max)) in TR-INF and TR-SHAM leveled off 65% above sedentary controls. In TR-SHAM, left and right ventricle weights were approximately 25% higher than in SED-SHAM, myocytes were approximately 13% longer; width remained unchanged. At physiological stimulation frequencies, relative myocyte shortening was markedly higher whereas peak systolic [Ca2+] and t(1/2) of Ca2+ transient decay were 10-20% lower, indicating higher Ca2+ sensitivity in cardiomyocytes from trained rats, compared to respective controls. In TR-INF the left and right ventricular weights, and myocyte length and width were 15, 23, 12, and 20% less than in SED-INF. Endurance training significantly increased the myocardial SR Ca2+ pump (SERCA-2) and sarcolemmal Na+-Ca2+-exchanger (NCX) protein levels to the extent that TR-INF did not differ from SED-SHAM.



This is the first study to show that aerobic endurance training attenuates the ventricular and cellular hypertrophy in failing hearts. Furthermore, training consistently restores contractile function, intracellular Ca2+ handling, and Ca2+-sensitivity in cardiomyocytes from rats with myocardial infarction.

title: Superior Cardiovascular Effect of Aerobic Interval Training Versus Moderate Continuous Training in Heart Failure Patients: A Randomized Study
abstract: Exercise training reduces the symptoms of chronic heart failure. Which exercise intensity yields maximal beneficial adaptations is controversial. Furthermore, the incidence of chronic heart failure increases with advanced age; it has been reported that 88% and 49% of patients with a first diagnosis of chronic heart failure are >65 and >80 years old, respectively. Despite this, most previous studies have excluded patients with an age >70 years. Our objective was to compare training programs with moderate versus high exercise intensity with regard to variables associated with cardiovascular function and prognosis in patients with postinfarction heart failure.  Twenty-seven patients with stable postinfarction heart failure who were undergoing optimal medical treatment, including &bgr;-blockers and angiotensin-converting enzyme inhibitors (aged 75.5±11.1 years; left ventricular [LV] ejection fraction 29%; &OV0312;o2peak 13 mL · kg−1 · min−1) were randomized to either moderate continuous training (70% of highest measured heart rate, ie, peak heart rate) or aerobic interval training (95% of peak heart rate) 3 times per week for 12 weeks or to a control group that received standard advice regarding physical activity. &OV0312;o2peak increased more with aerobic interval training than moderate continuous training (46% versus 14%, P<0.001) and was associated with reverse LV remodeling. LV end-diastolic and end-systolic volumes declined with aerobic interval training only, by 18% and 25%, respectively; LV ejection fraction increased 35%, and pro-brain natriuretic peptide decreased 40%. Improvement in brachial artery flow-mediated dilation (endothelial function) was greater with aerobic interval training, and mitochondrial function in lateral vastus muscle increased with aerobic interval training only. The MacNew global score for quality of life in cardiovascular disease increased in both exercise groups. No changes occurred in the control group.  Exercise intensity was an important factor for reversing LV remodeling and improving aerobic capacity, endothelial function, and quality of life in patients with postinfarction heart failure. These findings may have important implications for exercise training in rehabilitation programs and future studies.

title: Impact of Regular Physical Activity on the NAD(P)H Oxidase and Angiotensin Receptor System in Patients With Coronary Artery Disease
abstract: In patients with stable coronary artery disease, physical exercise training (ET) improves endothelial dysfunction. A potential mechanism mediating the enhanced vasomotor function is a reduced breakdown of endothelium-derived nitric oxide by reactive oxygen species (ROS). The aim of the present study was to analyze the impact of ET on sources of ROS generation in the left internal mammary artery of patients with symptomatic coronary artery disease. In left internal mammary artery rings sampled during bypass surgery from 45 patients randomized to either a training (n=22) or an inactive control (n=23) group, the mRNA expression of NAD(P)H oxidase subunits, NAD(P)H oxidase activity, and ROS production were assessed. In addition, endothelial function, expression of angiotensin II (Ang II) receptor type 1 and 2 (AT1-R and AT2-R), and Ang II-mediated vasoconstriction were determined. ET resulted in a significant lower expression of gp91phox (23.1±0.5 versus 69.1±18.1 arbitrary units, training versus control), p22phox (0.7±0.3 versus 2.0±0.5 arbitrary units), and Nox4 (2.7±1.2 versus 5.4±1.0 arbitrary units). Enzymatic activity (2.1±0.3 versus 4.9±0.4 mU/mg) and ROS generation (0.02±0.01 versus 0.06±0.02 arbitrary units) were significantly lower in the training compared with the control group. On a functional level, ET resulted in improved acetylcholine-mediated vasodilatation and a 49% reduction in Ang II–induced vasoconstriction, accompanied by lower AT1-R (3.7±0.8 versus 16.6±5.7 arbitrary units, training versus control) and higher AT2-R (7.8±2.5 versus 1.6±0.7 arbitrary units) mRNA expression. ET reduces vascular expression of NAD(P)H oxidase and AT1-R, resulting in decreased local ROS generation. These molecular effects converge in a reduced Ang II–mediated vasoconstriction.

title: Exercise Training in Patients With Advanced Chronic Heart Failure (NYHA IIIb) Promotes Restoration of Peripheral Vasomotor Function, Induction of Endogenous Regeneration, and Improvement of Left Ventricular Function
abstract: Attenuated peripheral perfusion in patients with advanced chronic heart failure (CHF) is partially the result of endothelial dysfunction. This has been causally linked to an impaired endogenous regenerative capacity of circulating progenitor cells (CPC). The aim of this study was to elucidate whether exercise training (ET) affects exercise intolerance and left ventricular (LV) performance in patients with advanced CHF (New York Heart Association class IIIb) and whether this is associated with correction of peripheral vasomotion and induction of endogenous regeneration. Thirty-seven patients with CHF (LV ejection fraction 24±2%) were randomly assigned to 12 weeks of ET or sedentary lifestyle (control). At the beginning of the study and after 12 weeks, maximal oxygen consumption (Vo2max) and LV ejection fraction were determined; the number of CD34+/KDR+ CPCs was quantified by flow cytometry and CPC functional capacity was determined by migration assay. Flow-mediated dilation was assessed by ultrasound. Capillary density was measured in skeletal muscle tissue samples. In advanced CHF, ET improved Vo2max by +2.7±2.2 versus −0.8±3.1 mL/min/kg in control (P=0.009) and LV ejection fraction by +9.4±6.1 versus −0.8±5.2% in control (P<0.001). Flow-mediated dilation improved by +7.43±2.28 versus +0.09±2.18% in control (P<0.001). ET increased the number of CPC by +83±60 versus −6±109 cells/mL in control (P=0.014) and their migratory capacity by +224±263 versus −12±159 CPC/1000 plated CPC in control (P=0.03). Skeletal muscle capillary density increased by +0.22±0.10 versus −0.02±0.16 capillaries per fiber in control (P<0.001). Twelve weeks of ET in patients with advanced CHF is associated with augmented regenerative capacity of CPCs, enhanced flow-mediated dilation suggestive of improvement in endothelial function, skeletal muscle neovascularization, and improved LV function.

title: Central adaptations to exercise training in patients with chronic heart failure
abstract: In chronic heart failure patients, the increase of peak VO2 observed after a period of aerobic training is currently attributed more to peripheral (skeletal muscle) than central (heart) adaptations. This paper reviews the current scientific evidence regarding the existence or the absence of significant training-induced adaptations of peak cardiac output and its determinants in patients with chronic heart failure due to left ventricular systolic dysfunction. It is concluded that, on the basis of available literature, a training-induced significant increase of peak cardiac output with respect to pre-training values does exist in the chronic heart failure population. Such an effect is due to adaptations of the main cardiac output determinants, that is, heart rate and stroke volume, whose relative contribution to the cardiac output increase will vary in the single patient due to variability in the individual response to the training stimulus. Moreover, these data emphasize both the safety and even the central morpho-functional benefits of aerobic training programs in the chronic heart failure population, setting the stage for an even more widespread use of this non-pharmacologic intervention in the everyday clinical practice. As most studies considered in this review were conducted in the pre-beta-blockers era, the possibility to extend the conclusions of this paper to chronic heart failure patients on beta-blocking therapy remains to be verified.

title: Aerobic interval training enhances cardiomyocyte contractility and Ca2+ cycling by phosphorylation of CaMKII and Thr-17 of phospholamban.
abstract: Cardiac adaptation to aerobic exercise training includes improved cardiomyocyte contractility and calcium handling. Our objective was to determine whether cytosolic calcium/calmodulin-dependent kinase II and its downstream targets are modulated by exercise training. A six-week aerobic interval training program by treadmill running increased maximal oxygen uptake by 35% in adult mice, whereupon left ventricular cardiomyocyte function was studied and myocardial tissue samples were used for biochemical analysis. Cardiomyocytes from trained mice had enhanced contractility and faster relaxation rates, which coincided with larger amplitude and faster decay of the calcium transient, but not increased peak systolic calcium levels. These changes were associated with reduced phospholamban expression relative to sarcoplasmic reticulum calcium ATPase and constitutively increased phosphorylation of phospholamban at the threonine 17, but not at the serine 16 site. Calcium/calmodulin-dependent kinase IIdelta phosphorylation was increased at threonine 287, indicating activation. To investigate the physiological role of calcium/calmodulin-dependent kinase IIdelta phosphorylation, this kinase was blocked specifically by autocamtide-2 related inhibitory peptide II. This maneuver completely abolished training-induced improvements of cardiomyocyte contractility and calcium handling and blunted, but did not completely abolish the training-induced increase in Ca(2+) sensitivity. Also, inhibition of calcium/calmodulin-dependent kinase II reduced the greater frequency-dependent acceleration of relaxation that was observed after aerobic interval training. These observations indicate that calcium/calmodulin-dependent kinase IIdelta contributes significantly to the functional adaptation of the cardiomyocyte to regular exercise training.

title: Running speed and maximal oxygen uptake in rats and mice: practical implications for exercise training
abstract: Valid and reliable experimental models are essential to gain insight into the cellular and molecular mechanisms underlying the beneficial effects of exercise in prevention, treatment, and rehabilitation of lifestyle-related diseases. Studies with large changes, low variation, and reproducible training outcome require individualized training intensity, controlled by direct measurements of maximal oxygen uptake or heart rate. As this approach is expensive and time consuming, we discuss whether maximal treadmill running speed in a gradually increasing ramp protocol might be sufficient to control intensity without lossing accuracy. Combined data from six studies of rats and mice from our lab demonstrated a close correlation between running speed and oxygen uptake. This relationship changed towards a steeper linear slope after endurance training, indicating improved work economy, that is, less oxygen was consumed at fixed submaximal running speeds. Maximal oxygen uptake increased 40-70% after high-intensity aerobic interval training in mice and rats. The speed at which oxygen uptake reached a plateau, increased in parallel with the change in maximal oxygen uptake during the training period. Although this suggests that running speed can be used to assess training intensity throughout a training program, the problem is to determine the exact relative intensity related to maximal oxygen uptake from running speed alone. We therefore suggest that directly measured oxygen uptake should be used to assess exercise intensity and optimize endurance training in rats and mice. Running speed may serve as a supplement to ensure this intensity.

title: Moderate vs. high exercise intensity: differential effects on aerobic fitness, cardiomyocyte contractility, and endothelial function.
abstract: Current guidelines are controversial regarding exercise intensity in cardiovascular prevention and rehabilitation. Although high-intensity training induces larger increases in fitness and maximal oxygen uptake (VO(2max)), moderate intensity is often recommended as equally effective. Controlled preclinical studies and randomized clinical trials are required to determine whether regular exercise at moderate versus high intensity is more beneficial. We therefore assessed relative effectiveness of 10-week HIGH versus moderate (MOD) exercise intensity on integrative and cellular functions.



Sprague-Dawley rats performed treadmill running intervals at either 85%-90% (HIGH) or 65%-70% (MOD) of VO2max 1 h per day, 5 days per week. Weekly VO2max-testing adjusted exercise intensity.



HIGH and MOD increased VO2max by 71% and 28%, respectively. This was paralleled by intensity-dependent cardiomyocyte hypertrophy, 14% and 5% in HIGH and MOD, respectively. Cardiomyocyte function (fractional shortening) increased by 45% and 23%, contraction rate decreased by 43% and 39%, and relaxation rate decreased by 20% and 10%, in HIGH and MOD, respectively. Ca2+ transient time-courses paralleled contraction/relaxation, whereas Ca2+ sensitivity increased 40% and 30% in HIGH and MOD, respectively. Carotid artery endothelial function improved similarly with both intensities. EC50 for acetylcholine-induced relaxation decreased 4.3-fold in HIGH (p < 0.05) and 2.8-fold in MOD (p < 0.20) as compared to sedentary; difference HIGH versus MOD 1.5-fold (p = 0.72). Multiple regression identified rate of systolic Ca2+ increase and diastolic myocyte relengthening as main variables associated with VO2max. Cell hypertrophy, contractility and vasorelaxation also correlated significantly with VO2max.



The present study demonstrates that cardiovascular adaptations to training are intensity-dependent. A close correlation between VO2max, cardiomyocyte dimensions and contractile capacity suggests significantly higher benefit with high intensity, whereas endothelial function appears equivalent at moderate levels. Thus, exercise intensity emerges as an important variable in future preclinical and clinical investigations.

title: A uniform enzymatic method for dissociation of myocytes from hearts and stomachs of vertebrates.
abstract: A method is presented that consistently yields a large number of calcium-tolerant myocytes from mammalian, amphibian, and elasmobranch hearts and from mammalian stomach. The use of incubating solutions or cell harvesting techniques was not required. The time needed to isolate cells was shorter than previously reported values. Action potentials recorded from each cell type appear similar in configuration to that of the intact multicellular tissue. The isolated myocytes appear to tolerate long periods of electrophysiological experimentation using the "giga-seal" suction electrode technique of Hamill et al. ( 85-100, 1981). This method is ideally suited for comparative electrophysiological studies, since the procedure for cell isolation was not seriously modified according to the preparation or species used.

title: Design of the mammalian respiratory system. III Scaling maximum aerobic capacity to body mass: wild and domestic mammals.
abstract: The purpose of this study was to determine whether the maximal rate of oxygen consumption (Vo2max) is scaled proportionally to Mb1.0, as the diffusing capacity of the lung, or proportionally to Mb0.75, as the standardized resting rate of oxygen consumption (V02std). We measured Vo2max on a variety of mammalian species (14 wild species and 8 domestic or laboratory species ranging in Mb from 7.2 g to 263 kg) using the same 'treadmill' procedure for all animals. For the wild species we found: Vo2max = 1.94.Mb0.79; r=0.995 where Vo2max has the units ml . sec-1 and mb is in kg. There was a great variability in Vo2max among domestic species of the same size, horse and dog having a Vo2max more than 3 times that of a cow and sheep, respectively. Both the variability in Vo2max with body size and among animals of the same size provide powerful tools for investigating the relationship between structure and function at each step in the respiratory system, from the oxygen in environmental air to the oxygen sink in the mitochondria.

title: Beta-adrenergic receptor signaling in the heart: role of CaMKII.
abstract: The multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) targets a number of Ca(2+) homeostatic proteins and regulates gene transcription. Many of the substrates phosphorylated by CaMKII are also substrates for protein kinase A (PKA), the best known downstream effector of beta-adrenergic receptor (beta-AR) signaling. While PKA and CaMKII are conventionally considered to transduce signals through separate pathways, there is a body of evidence suggesting that CaMKII is activated in response to beta-AR stimulation and that some of the downstream effects of beta-AR stimulation are actually mediated by CaMKII. The signaling pathway through which beta-AR stimulation activates CaMKII, in parallel with or downstream of PKA, is not well-defined. This review considers the evidence for and mechanisms by which CaMKII is activated in response to beta-AR stimulation. In addition the potential role of CaMKII in beta-AR regulation of cardiac function is considered. Notably, although many CaMKII targets (e.g., phospholamban or the ryanodine receptor) are central to the regulation of Ca(2+) handling, and effects of CaMKII on Ca(2+) handling are detectable, inhibition or gene deletion of CaMKII has relatively little effect on the acute physiological contractile response to beta-AR. On the other hand CaMKII expression and activity are increased in heart failure, a pathophysiological condition characterized by chronic stimulation of cardiac beta-ARs. Blockade of beta-ARs is an accepted therapy for treatment of chronic heart failure although the rationale for its beneficial effects in cardiomyocytes is uncertain. There is growing evidence that inhibition or gene deletion of CaMKII also has a significant beneficial impact on the development of heart failure. The possibility that excessive beta-AR stimulation is detrimental because of its effects on CaMKII mediated Ca(2+) handling disturbances (e.g., ryanodine receptor phosphorylation and diastolic SR Ca(2+) leak) is an intriguing hypothesis that merits future consideration.

title: Limiting factors for maximum oxygen uptake and determinants of endurance performance.
abstract: In the exercising human, maximal oxygen uptake (VO2max) is limited by the ability of the cardiorespiratory system to deliver oxygen to the exercising muscles. This is shown by three major lines of evidence: 1) when oxygen delivery is altered (by blood doping, hypoxia, or beta-blockade), VO2max changes accordingly; 2) the increase in VO2max with training results primarily from an increase in maximal cardiac output (not an increase in the a-v O2 difference); and 3) when a small muscle mass is overperfused during exercise, it has an extremely high capacity for consuming oxygen. Thus, O2 delivery, not skeletal muscle O2 extraction, is viewed as the primary limiting factor for VO2max in exercising humans. Metabolic adaptations in skeletal muscle are, however, critical for improving submaximal endurance performance. Endurance training causes an increase in mitochondrial enzyme activities, which improves performance by enhancing fat oxidation and decreasing lactic acid accumulation at a given VO2. VO2max is an important variable that sets the upper limit for endurance performance (an athlete cannot operate above 100% VO2max, for extended periods). Running economy and fractional utilization of VO2max also affect endurance performance. The speed at lactate threshold (LT) integrates all three of these variables and is the best physiological predictor of distance running performance.

title: The Molecular Bases of Training Adaptation
abstract: Skeletal muscle is a malleable tissue capable of altering the type and amount of protein in response to disruptions to cellular homeostasis. The process of exerciseinduced adaptation in skeletal muscle involves a multitude of signalling mechanisms initiating replication of specific DNA genetic sequences, enabling subsequent translation of the genetic message and ultimately generating a series of amino acids that form new proteins. The functional consequences of these adaptations are determined by training volume, intensity and frequency, and the half-life of the protein. Moreover, many features of the training adaptation are specific to the type of stimulus, such as the mode of exercise. Prolonged endurance training elicits a variety of metabolic and morphological changes, including mitochondrial biogenesis, fast-to-slow fibre-type transformation and substrate metabolism. In contrast, heavy resistance exercise stimulates synthesis of contractile proteins responsible for muscle hypertrophy and increases in maximal contractile force output. Concomitant with the vastly different functional outcomes induced by these diverse exercise modes, the genetic and molecular mechanisms of adaptation are distinct. With recent advances in technology, it is now possible to study the effects of various training interventions on a variety of signalling proteins and early-response genes in skeletal muscle. Although it cannot presently be claimed that such scientific endeavours have influenced the training practices of elite athletes, these new and exciting technologies have provided insight into how current training techniques result in specific muscular adaptations, and may ultimately provide clues for future and novel training methodologies. Greater knowledge of the mechanisms and interaction of exercise-induced adaptive pathways in skeletal muscle is important for our understanding of the aetiology of disease, maintenance of metabolic and functional capacity with aging, and training for athletic performance. This article highlights the effects of exercise on molecular and genetic mechanisms of training adaptation in skeletal muscle.

title: Divergent cell signaling after short-term intensified endurance training in human skeletal muscle.
abstract: Endurance training represents one extreme in the continuum of skeletal muscle plasticity. The molecular signals elicited in response to acute and chronic exercise and the integration of multiple intracellular pathways are incompletely understood. We determined the effect of 10 days of intensified cycle training on signal transduction in nine inactive males in response to a 1-h acute bout of cycling at the same absolute workload (164 +/- 9 W). Muscle biopsies were taken at rest and immediately and 3 h after the acute exercise. The metabolic signaling pathways, including AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), demonstrated divergent regulation by exercise after training. AMPK phosphorylation increased in response to exercise ( approximately 16-fold; P < 0.05), which was abrogated posttraining (P < 0.01). In contrast, mTOR phosphorylation increased in response to exercise ( approximately 2-fold; P < 0.01), which was augmented posttraining (P < 0.01) in the presence of increased mTOR expression (P < 0.05). Exercise elicited divergent effects on mitogen-activated protein kinase (MAPK) pathways after training, with exercise-induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation being abolished (P < 0.01) and p38 MAPK maintained. Finally, calmodulin kinase II (CaMKII) exercise-induced phosphorylation and activity were maintained (P < 0.01), despite increased expression ( approximately 2-fold; P < 0.05). In conclusion, 10 days of intensified endurance training attenuated AMPK, ERK1/2, and mTOR, but not CaMKII and p38 MAPK signaling, highlighting molecular pathways important for rapid functional adaptations and maintenance in response to intensified endurance exercise and training.

title: Early Exercise Training Normalizes Myofilament Function and Attenuates Left Ventricular Pump Dysfunction in Mice With a Large Myocardial Infarction
abstract: The extent and mechanism of the cardiac benefit of early exercise training following myocardial infarction (MI) is incompletely understood, but may involve blunting of abnormalities in Ca2+-handling and myofilament function. Consequently, we investigated the effects of 8-weeks of voluntary exercise, started early after a large MI, on left ventricular (LV) remodeling and dysfunction in the mouse. Exercise had no effect on survival, MI size or LV dimensions, but improved LV fractional shortening from 8±1 to 12±1%, and LVdP/dtP30 from 5295±207 to 5794±207 mm Hg/s (both P<0.05), and reduced pulmonary congestion. These global effects of exercise were associated with normalization of the MI-induced increase in myofilament Ca2+-sensitivity (&Dgr;pCa50=0.037). This effect of exercise was PKA-mediated and likely because of improved &bgr;1-adrenergic signaling, as suggested by the increased &bgr;1-adrenoceptor protein (48%) and cAMP levels (36%; all P<0.05). Exercise prevented the MI-induced decreased maximum force generating capacity of skinned cardiomyocytes (Fmax increased from 14.3±0.7 to 18.3±0.8 kN/m2P<0.05), which was associated with enhanced shortening of unloaded intact cardiomyocytes (from 4.1±0.3 to 7.0±0.6%; P<0.05). Furthermore, exercise reduced diastolic Ca2+-concentrations (by ∼30%, P<0.05) despite the unchanged SERCA2a and PLB expression and PLB phosphorylation status. Importantly, exercise had no effect on Ca2+-transient amplitude, indicating that the improved LV and cardiomyocyte shortening were principally because of improved myofilament function. In conclusion, early exercise in mice after a large MI has no effect on LV remodeling, but attenuates global LV dysfunction. The latter can be explained by the exercise-induced improvement of myofilament function.

title: Exercise training increases the Ca(2+) sensitivity of tension in rat cardiac myocytes.
abstract: The heart is known to respond to a program of chronic exercise in ways that enhance cardiac function. However, the cellular mechanisms involved in training-induced improvements in the contractile function of the myocardium are not known. In this study we tested the hypothesis that increased contractility of the myocardium associated with exercise training is due, in part, to increases in the Ca(2+) sensitivity of steady-state tension. Female Sprague-Dawley rats were randomly divided into sedentary control (C) and exercise-trained (T) groups. The T rats underwent 11 wk of progressive treadmill exercise (1 h/day, 5 days/wk, 26 m/min, 20% grade). Evidence of training effect included a 5.9% increase in heart mass, increases in heart weight-to-body weight ratio, and a 60% increase in skeletal muscle citrate synthase activity in T rats compared with C rats. After the training program, cardiac myocytes were isolated from T and C hearts. Myocytes were chemically skinned (i.e., the sarcolemma was removed) and attached to a force transducer, and steady-state tension was determined in solutions of various Ca(2+) concentrations ([Ca(2+)]). Myocytes isolated from the hearts of T rats showed a significantly (P < 0.01) increased sensitivity of tension to [Ca(2+)]. The [Ca(2+)] giving 50% of maximal tension (pCa(50)) was 5.90 +/- 0.033 and 5.82 +/- 0.023 (SD) in T and C myocytes, respectively (n = 70 myocytes/group). This result suggests that exercise training affects the myofibrillar proteins, such that Ca(2+) sensitivity is increased, and that this may be the mechanism that underlies, at least in part, the effect of training to increase myocardial contractility.

title: CaMKII inhibition protects against necrosis and apoptosis in irreversible ischemia-reperfusion injury.
abstract: Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the regulation of cardiac excitation-contraction coupling (ECC) as well as in apoptotic signaling and adverse remodeling. The goal of the present study is to investigate the role of CaMKII in irreversible ischemia and reperfusion (I/R) injury.



Isovolumic Langendorff perfused rat hearts were subjected to global no-flow I/R (45 min/120 min), and isolated myocytes were subjected to a protocol of simulated I/R (45 min simulated ischemia/60 min reoxygenation) either in the absence or presence of CaMKII inhibition [KN-93 (KN) or the CaMKII inhibitory peptide (AIP)].



In I/R hearts, an increase in CaMKII activity at the beginning of reperfusion was confirmed by the significantly increased phosphorylation of the Thr(17) site of phospholamban. In the presence of KN, contractile recovery at the end of reperfusion was almost double that of I/R hearts. This recovery was associated with a significant decrease in the extent of infarction, lactate dehydrogenase release (necrosis), TUNEL-positive cells, caspase-3 activity, and an increase in the Bcl-2/Bax ratio (apoptosis). In isolated myocytes, both KN and AIP prevented simulated I/R-induced spontaneous contractile activity and cell mortality. Similar results were obtained when inhibiting the reverse mode Na(+)/Ca(2+) exchanger (NCX) with KB-R7943, sarcoplasmic reticulum (SR) function with ryanodine and thapsigargin, or SR Ca(2+) release with tetracaine. In contrast, overexpression of CaMKII decreased cell viability from 52+/-3% to 26+/-2%.



Taken together, the present findings are the first to establish CaMKII as a fundamental component of a cascade of events integrating the NCX, the SR, and mitochondria that promote cellular apoptosis and necrosis in irreversible I/R injury.

title: Calmodulin Kinase II and Arrhythmias in a Mouse Model of Cardiac Hypertrophy
abstract: Calmodulin kinase (CaMK) II is linked to arrhythmia mechanisms in cellular models where repolarization is prolonged. CaMKII upregulation and prolonged repolarization are general features of cardiomyopathy, but the role of CaMKII in arrhythmias in cardiomyopathy is unknown. We studied a mouse model of cardiac hypertrophy attributable to transgenic (TG) overexpression of a constitutively active form of CaMKIV that also has increased endogenous CaMKII activity. ECG-telemetered TG mice had significantly more arrhythmias than wild-type (WT) littermate controls at baseline, and arrhythmias were additionally increased by isoproterenol. Arrhythmias were significantly suppressed by an inhibitory agent targeting endogenous CaMKII. TG mice had longer QT intervals and action potential durations than WT mice, and TG cardiomyocytes had frequent early afterdepolarizations (EADs), a hypothesized mechanism for triggering arrhythmias. EADs were absent in WT cells before and after isoproterenol, whereas EAD frequency was unaffected by isoproterenol in TG mice. L-type Ca2+ channels (LTTCs) can activate EADs, and LTCC opening probability (Po) was significantly higher in TG than WT cardiomyocytes before and after isoproterenol. A CaMKII inhibitory peptide equalized TG and WT LTCC Po and eliminated EADs, whereas a peptide antagonist of the Na+/Ca2+ exchanger current, also hypothesized to support EADs, was ineffective. These findings support the hypothesis that CaMKII is a proarrhythmic signaling molecule in cardiac hypertrophy in vivo. Cellular studies point to EADs as a triggering mechanism for arrhythmias but suggest that the increase in arrhythmias after &bgr;-adrenergic stimulation is independent of enhanced EAD frequency.

title: Mechanisms and potential therapeutic targets for ventricular arrhythmias associated with impaired cardiac calcium cycling.
abstract: The close relationship between life-threatening ventricular arrhythmias and contractile dysfunction in the heart implicates intracellular calcium cycling as an important underlying mechanism of arrhythmogenesis. Despite this close association, however, the mechanisms of arrhythmogenesis attributable to impaired calcium cycling are not fully appreciated or understood. In this report we review some of the current thinking regarding arrhythmia mechanisms associated with either abnormal impulse initiation (i.e. arrhythmia triggers) or impulse propagation (i.e. arrhythmia substrates). In all cases, the mechanisms are primarily related to dysfunction of calcium regulatory proteins associated with the sarcomere. These findings highlight the broad scope of arrhythmias associated with abnormal calcium cycling, and provide a basis for a causal relationship between cardiac electrical instability and contractile dysfunction. Moreover, calcium cycling proteins may provide much needed targets for novel antiarrhythmic therapies.

title: Calmodulin Kinase II Inhibition Shortens Action Potential Duration by Upregulation of K+ Currents
abstract: The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by elevated intracellular Ca2+ (Ca2+i), and mice with chronic myocardial CaMKII inhibition (Inh) resulting from transgenic expression of a CaMKII inhibitory peptide (AC3-I) unexpectedly showed action potential duration (APD) shortening. Inh mice exhibit increased L-type Ca2+ current (ICa), because of upregulation of protein kinase A (PKA) activity, and decreased CaMKII-dependent phosphorylation of phospholamban (PLN). We hypothesized that CaMKII is a molecular signal linking Ca2+i to repolarization. Whole cell voltage-clamp recordings revealed that the fast transient outward current (Ito,f) and the inward rectifier current (IK1) were selectively upregulated in Inh, compared with wild-type (WT) and transgenic control, mice. Breeding Inh mice with mice lacking PLN returned Ito,f and IK1 to control levels and equalized the APD and QT intervals in Inh mice to control and WT levels. Dialysis of AC3-I into WT cells did not result in increased Ito,f or IK1, suggesting that enhanced cardiac repolarization in Inh mice is an adaptive response to chronic CaMKII inhibition rather than an acute effect of reduced CaMKII activity. Increasing PKA activity, by cell dialysis with cAMP, or inhibition of PKA did not affect IK1 in WT cells. Dialysis of WT cells with cAMP also reduced Ito,f, suggesting that PKA upregulation does not increase repolarizing K+ currents in Inh mice. These findings provide novel in vivo and cellular evidence that CaMKII links Ca2+i to cardiac repolarization and suggest that PLN may be a critical CaMKII target for feedback regulation of APD in ventricular myocytes.

title: Calmodulin kinase II inhibition protects against myocardial cell apoptosis in vivo.
abstract: Inhibition of the multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) or depletion of sarcoplasmic reticulum (SR) Ca(2+) stores protects against apoptosis from excessive isoproterenol (Iso) stimulation in cultured ventricular myocytes, suggesting that CaMKII inhibition could be a novel approach to reducing cell death in conditions of increased adrenergic tone, such as myocardial infarction (MI), in vivo. We used mice with genetic myocardial CaMKII inhibition due to transgenic expression of a highly specific CaMKII inhibitory peptide (AC3-I) to test whether CaMKII was important for apoptosis in vivo. A second line of mice expressed a scrambled, inactive form of AC3-I (AC3-C). AC3-C and wild-type (WT) littermates were used as controls. AC3-I mice have reduced SR Ca(2+) content and are resistant to Iso- and MI-induced apoptosis compared with AC3-C and WT mice. Phospholamban (PLN) is a target for modulation of SR Ca(2+) content by CaMKII. PLN(-/-) mice have increased susceptibility to Iso-induced apoptosis. Verapamil pretreatment prevented Iso-induced apoptosis in PLN(-/-) mice, indicating the involvement of a Ca(2+)-dependent pathway. AC3-I and AC3-C mice were bred into a PLN(-/-) background. Loss of PLN increased and equalized SR Ca(2+) content in AC3-I, AC3-C, and WT mice and abolished the resistance to apoptosis in AC3-I mice after MI. There was a trend (P = 0.07) for increased Iso-induced apoptosis in AC3-I mice lacking PLN compared with AC3-I mice with PLN. These findings indicate CaMKII is proapoptotic in vivo and suggest that regulation of SR Ca(2+) content by PLN contributes to the antiapoptotic mechanism of CaMKII inhibition.

title: The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells.
abstract: We reported that one of the isoquinolinesulfonamide derivatives, KN-62, is a potent and specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Tokumitsu, H., Chijiwa, T., Hagiwara, M., Mizutani, A., Terasawa, M. and Hidaka, H. (1990) ). We have now investigated the inhibitory property of a newly synthesized methoxybenzenesulfonamide, KN-93, on CaMKII activity in situ and in vitro. KN-93 elicited potent inhibitory effects on CaMKII phosphorylating activity with an inhibition constant of 0.37 microM but this compound had no significant effects on the catalytic activity of cAMP-dependent protein kinase, Ca2+/phospholipid dependent protein kinase, myosin light chain kinase and Ca(2+)-phosphodiesterase. KN-93 also inhibited the autophosphorylation of both the alpha- and beta-subunits of CaMKII. Kinetic analysis indicated that KN-93 inhibits CaMKII, in a competitive fashion against calmodulin. To evaluate the regulatory role of CaMKII on catecholamine metabolism, we examined the effect of KN-93 on dopamine (DA) levels in PC12h cells. The DA levels decreased in the presence of KN-93. Further, the tyrosine hydroxylase (TH) phosphorylation induced by KCl or acetylcholine was significantly suppressed by KN-93 in PC12h cells while events induced by forskolin or 8-Br-cAMP were not affected. These results suggest that KN-93 inhibits DA formation by modulating the reaction rate of TH to reduce the Ca(2+)-mediated phosphorylation levels of the TH molecule.

title: CaMKII-independent effects of KN93 and its inactive analog KN92: reversible inhibition of L-type calcium channels.
abstract: Widely regarded as a specific and potent inhibitor of CaM kinases, especially CaMKII, KN93 has long been used to investigate the possible roles of CaMKII in a wide range of biological functions and systems, such as cultured cells, primary neurons, and brain slices. However, here we present evidence showing that KN93 and its structural analog KN92, which does not inhibit CaMKII, exert an unexpected, reversible, and specific reduction of currents of L-type calcium channels (CaV1.3 and CaV1.2), as compared to N-type calcium channels (CaV2.2). This effect is dependent not only on incubation time, but also on the dose of KN93 or KN92. Moreover, the effect appears to be independent of endocytosis, exocytosis, and proteasome activity. Washout and return to normal media rescues the L channel currents. Conversely, the structurally unrelated CaMKII inhibitor, AIP, fails to mimic the KN93/KN92 effect on L channel currents. Together, our data suggest that, in addition to inhibiting CaMKII, KN93 also affects CaV1.3 and CaV1.2 calcium channels in a CaMKII-independent manner.

title: KN-93 (2-[N-(2-Hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), a Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor, Is a Direct Extracellular Blocker of Voltage-Gated Potassium Channels
abstract: The effect of Ca2+/calmodulin-dependent protein kinase II (CaMK II) on voltage-gated ion channels is widely studied through the use of specific CaMK II blockers such as 2-[N-(2-hydroxyethyl)]-N-(4methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN-93). The present study demonstrates that KN-93 is a direct extracellular blocker of a wide range of cloned Kv channels from a number of different subfamilies. In all channels tested, the effect of 1 μM KN-93 was independent of CaMK II because 1 μM2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, phosphate (KN-92), an inactive analog of KN-93, caused similar inhibition of currents. In addition, dialysis of cells with 10 μM CaMK II inhibitory peptide fragment 281-301 (CIP) had no effect on current kinetics and did not prevent the inhibitory effect of KN-93. The IC50 for block of the Kv1.5 channel (used as an example to determine the nature of KN-93 block) was 307 ± 12 nM. KN-93 blocked open channels with little voltage dependence that did not alter the V1/2 of channel activation. Removal of P/C-type inactivation by mutation of arginine 487 to valine in the outer pore region of Kv1.5 (R487V) greatly reduced KN-93 block, whereas enhancement of inactivation induced by mutation of threonine 462 to cysteine (T462C) increased the potency of KN-93 by 4-fold. This suggested that KN-93 acted through promotion and stabilization of C-type inactivation. Importantly, KN-93 was ineffective as a blocker when applied intracellularly, suggesting that CaMK II-independent effects of KN-93 on Kv channels can be circumvented by intracellular application of KN-93.

title: Inhibitors of calmodulin-dependent protein kinase are nonspecific blockers of voltage-dependent K+ channels in vascular myocytes.
abstract: The present study was undertaken to investigate the effects of specific inhibitors of calmodulin-dependent protein kinase II (CamKII) on macroscopic voltage-dependent K(+) current (K(V)) recorded from rabbit portal vein smooth muscle cells. Inhibition of L-type Ca(2+) current facilitation by 1 microM KN-62, a blocker of CamKII, was first demonstrated and provided evidence for functional CamKII activity in this preparation. KN-93, another specific and more potent inhibitor of CamKII in the rat brain, suppressed K(V) and enhanced the rate of inactivation in a dose-dependent manner, in cells dialyzed with both low (0.1 mM) and high (10 mM) EGTA pipette solution. Prolonged dialysis with 10 microM of a synthetic peptide inhibitor of CamKII (fragment 281-301) had little effect on K(V) and did not prevent the inhibitory action of KN-93 on the current. The estimated IC(50) for inhibiting peak and late currents during 250-ms steps to +60 mV (holding potential = -60 mV) were 2.9 and 0.27 microM, respectively. KN-93 also induced slight shifts of the steady-state activation (-7 mV) and inactivation (-6 mV) curves. KN-62, and KN-92, an inactive analog of KN-93, produced effects similar to those of KN-93. In current clamp experiments, 5 microM KN-93 depolarized the myocytes from a control resting membrane potential of -42.3 +/- 2.8 mV to -28.5 +/- 1.4 mV, an effect that was partially reversible after washout (-34.4 +/- 1.3 mV, n = 6). In conclusion, blockers of CamKII produce nonspecific inhibitory effects on K(V) that warrant cautious use of these compounds in physiological experiments designed to assess the role of CamKII.

title: The Nuclear δB Isoform of Ca2+/Calmodulin-dependent Protein Kinase II Regulates Atrial Natriuretic Factor Gene Expression in Ventricular Myocytes*
abstract: Cultured neonatal ventricular myocytes display features of myocardial hypertrophy including increased cell size, myofilament organization, and reexpression of the embryonic gene for atrial natriuretic factor (ANF). KN-93, an inhibitor of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase II), blocked the induction of these responses by the α1-adrenergic receptor agonist phenylephrine, whereas its inactive analog KN-92 did not. To directly determine whether CaM kinase II could regulate ANF gene expression, we transiently expressed each of three isoforms of CaM kinase II (α, δB, and δC) along with an ANF promoter/luciferase reporter gene. The δB isoform markedly increased luciferase gene expression, whereas comparable levels of the δC and α isoforms were ineffective. Expression of δB-CaM kinase II also potentiated phenylephrine-mediated ANF gene expression, and this effect was blocked by KN-93 but not by KN-92. The ability of δB-CaM kinase II to transactivate a truncated ANF promoter, containing a serum response element (SRE) required for phenylephrine-inducible gene expression, was lost when this SRE was mutated. The δB isoform of CaM kinase II has been shown to exhibit nuclear localization. Coexpression of the non-nuclear δC or α isoforms, which can form multimers with the δB isoform, prevented the nuclear localization of δB-CaM kinase II and also blocked its effects on ANF reporter gene and protein expression. In addition, a chimeric α-CaM kinase II which contains the nuclear localization signal of the δB isoform was able to induce ANF reporter gene expression, albeit to a lesser extent than δB-CaM kinase II. These data are the first to assign a function to the δB isoform of CaM kinase II and to link its nuclear localization to subsequent activation of cardiac gene expression.

title: Ca2+/Calmodulin-dependent Kinase II and Calcineurin Play Critical Roles in Endothelin-1-induced Cardiomyocyte Hypertrophy*
abstract: Endothelin-1 (ET-1) induces cardiac hypertrophy. Because Ca2+ is a major second messenger of ET-1, the role of Ca2+ in ET-1-induced hypertrophic responses in cultured cardiac myocytes of neonatal rats was examined. ET-1 activated the promoter of the β-type myosin heavy chain gene (β-MHC) (−354 to +34 base pairs) by about 4-fold. This activation was inhibited by chelation of Ca2+ and the blocking of protein kinase C activity. Similarly, the β-MHC promoter was activated by Ca2+ionophores and a protein kinase C activator. β-MHCpromoter activation induced by ET-1 was suppressed by pretreatment with the calmodulin inhibitor, W7, the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor, KN62, and the calcineurin inhibitor, cyclosporin A. β-MHC promoter activation by ET-1 was also attenuated by overexpression of dominant-negative mutants of CaMKII and calcineurin. ET-1 increased the activity of CaMKII and calcineurin in cardiac myocytes. Pretreatment with KN62 and cyclosporin A strongly suppressed ET-1-induced increases in [3H]phenylalanine uptake and in cell size. These results suggest that Ca2+ plays a critical role in ET-1-induced cardiomyocyte hypertrophy by activating CaMKII- and calcineurin-dependent pathways.