Yoichiro Kusakari

p38α Mitogen-Activated Protein Kinase Plays a Critical Role in Cardiomyocyte Survival but Not in Cardiac Hypertrophic Growth in Response to Pressure Overload

ABSTRACT The molecular mechanism for the transition from cardiac hypertrophy, an adaptive response to biomechanical stress, to heart failure is poorly understood. The mitogen-activated protein kinase p38α is a key component of stress response pathways in various types of cells. In this study, we attempted to explore the in vivo physiological functions of p38α in hearts. First, we generated mice with floxed p38α alleles and crossbred them with mice expressing the Cre recombinase under the control of the α-myosin heavy-chain promoter to obtain cardiac-specific p38α knockout mice. These cardiac-specific p38α knockout mice were born normally, developed to adulthood, were fertile, exhibited a normal life span, and displayed normal global cardiac structure and function. In response to pressure overload to the left ventricle, they developed significant levels of cardiac hypertrophy, as seen in controls, but also developed cardiac dysfunction and heart dilatation. This abnormal response to pressure overload was accompanied by massive cardiac fibrosis and the appearance of apoptotic cardiomyocytes. These results demonstrate that p38α plays a critical role in the cardiomyocyte survival pathway in response to pressure overload, while cardiac hypertrophic growth is unaffected despite its dramatic down-regulation.

Targeted deletion of apoptosis signal-regulating kinase 1 attenuates left ventricular remodeling

Left ventricular remodeling that occurs after myocardial infarction (MI) and pressure overload is generally accepted as a determinant of the clinical course of heart failure. The molecular mechanism of this process, however, remains to be elucidated. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays an important role in stress-induced apoptosis. We used ASK1 knockout mice (ASK-/-) to test the hypothesis that ASK1 is involved in development of left ventricular remodeling. ASK-/- hearts showed no morphological or histological defects. Echocardiography and cardiac catheterization revealed normal global structure and function. Left ventricular structural and functional remodeling were determined 4 weeks after coronary artery ligation or thoracic transverse aortic constriction (TAC). ASK-/- had significantly smaller increases in left ventricular end-diastolic and end-systolic ventricular dimensions and smaller decreases in fractional shortening in both experimental models compared with WT mice. The number of terminal deoxynucleotidyl transferase biotin-dUDP nick end-labeling-positive myocytes after MI or TAC was decreased in ASK-/- compared with that in WT mice. Overexpression of a constitutively active mutant of ASK1 induced apoptosis in isolated rat neonatal cardiomyocytes, whereas neonatal ASK-/- cardiomyocytes were resistant to H2O2-induced apoptosis. An in vitro kinase assay showed increased ASK1 activity in heart after MI or TAC in WT mice. Thus, ASK1 plays an important role in regulating left ventricular remodeling by promoting apoptosis.

Cardiac-specific overexpression of a high Ca2+ affinity mutant of SERCA2a attenuates in vivo pressure overload cardiac hypertrophy

In cardiomyocytes, calcium plays important roles as a signal in cardiac hypertrophy and contraction‐relaxation cycling. Elevation of Ca2+ concentration in myoplasm is associated with the onset and progression of hypertrophy as well as the enhancement of contractility. The cardiac Ca2+ ATPase (SERCA2a) of the sarcoplasmic reticulum plays a dominant role in lowering cytoplasmic calcium levels during relaxation and is regulated by phospholamban (PLN). To examine whether the modulation of SERCA2a activity results in the attenuation of cardiac hypertrophy and enhancement of contractility, we generated transgenic mice (TG) overexpressing a high calcium affinity SERCA2a mutant (K397/400E), lacking a functional association with PLN. In the TG hearts, the apparent affinity of SERCA2a for Ca2+ significantly increased compared with their nontransgenic littermate controls. The TG showed increased contraction and relaxation, with increases in the amplitude of Ca2+ transient and rapid Ca2+ decay. Upon induction of pressure overload by transverse aortic constriction, the TG developed less cardiac hypertrophy than littermate controls did. The activation of Ca2+‐sensitive protein kinase C by pressure overload was significantly attenuated in the TG hearts. Our findings indicate an association of SERCA2a activity with cardiac hypertrophy and thus a new therapeutic target for the prevention and treatment of cardiac hypertrophy.

Interaction of α1-Adrenoceptor Subtypes With Different G Proteins Induces Opposite Effects on Cardiac L-type Ca2+ Channel

We examined the effect of &agr;1-adrenoceptor subtype-specific stimulation on L-type Ca2+ current (ICa) and elucidated the subtype-specific intracellular mechanisms for the regulation of L-type Ca2+ channels in isolated rat ventricular myocytes. We confirmed the protein expression of &agr;1A- and &agr;1B-adrenoceptor subtypes at the transverse tubules (T-tubules) and found that simultaneous stimulation of these 2 receptor subtypes by nonsubtype selective agonist, phenylephrine, showed 2 opposite effects on ICa (transient decrease followed by sustained increase). However, selective &agr;1A-adrenoceptor stimulation (≥0.1 &mgr;mol/L A61603) only potentiated ICa, and selective &agr;1B-adrenoceptor stimulation (10 &mgr;mol/L phenylephrine with 2 &mgr; mol/L WB4101) only decreased ICa. The positive effect by &agr;1A-adrenoceptor stimulation was blocked by the inhibition of phospholipase C (PLC), protein kinase C (PKC), or Ca2+/calmodulin-dependent protein kinase II (CaMKII). The negative effect by &agr;1B-adrenoceptor stimulation disappeared after the treatment of pertussis toxin or by the prepulse depolarization, but was not attriburable to the inhibition of cAMP-dependent pathway. The translocation of PKC&dgr; and ϵ to the T-tubules was observed only after &agr;1A-adrenoceptor stimulation, but not after &agr;1B-adrenoceptor stimulation. Immunoprecipitaion analysis revealed that &agr;1A-adrenoceptor was associated with Gq/11, but &agr;1B-adrenoceptor interacted with one of the pertussis toxin-sensitive G proteins, Go. These findings demonstrated that the interactions of &agr;1-adrenoceptor subtypes with different G proteins elicit the formation of separate signaling cascades, which produce the opposite effects on ICa. The coupling of &agr;1A-adrenoceptor with Gq/11-PLC-PKC-CaMKII pathway potentiates ICa. In contrast, &agr;1B-adrenoceptor interacts with Go, of which the &bgr;&ggr;-complex might directly inhibit the channel activity at T-tubules.

Presenilin 2 regulates the systolic function of heart by modulating Ca2+ signaling

Genetic studies of families with familial Alzheimers disease have implicated presenilin 2 (PS2) in the pathogenesis of this disease. PS2 is ubiquitously expressed in various tissues including hearts. In this study, we examined cardiac phenotypes of PS2 knockout (PS2KO) mice to elucidate a role of PS2 in hearts. PS2KO mice developed normally with no evidence of cardiac hypertrophy and fibrosis. Invasive hemodynamic analysis revealed that cardiac contractility in PS2KO mice increased compared with that in their littermate controls. A study of isolated papillary muscle showed that peak amplitudes of Ca2+ transients and peak tension were significantly higher in PS2KO mice than those in their littermate controls. PS2KO mouse hearts exhibited no change in expression of calcium regulatory proteins. Since it has been demonstrated that PS2 in brain interacts with sorcin, which serves as a modulator of cardiac ryanodine receptor (RyR2), we tested whether PS2 also interacts with RyR2. Immmunoprecipitation analysis showed that PS2, sorcin, and RyR2 interact with each other in HEK‐293 cells overexpressing these proteins or in mouse hearts. Immunohistochemistry of heart muscle indicated that PS2 colocalizes with RyR2 and sorcin at the Z‐lines. Elevated Ca2+ attenuated the association of RyR2 with PS2, whereas the association of sorcin with PS2 was enhanced. The enhanced Ca2+ transients and contractility in PS2KO mice were observed at low extracellular [Ca2+] but not at high levels of [Ca2+]. Taken together, our results suggest that PS2 plays an important role in cardiac excitation‐contraction coupling by interacting with RyR2.

Bupivacaine attenuates contractility by decreasing sensitivity of myofilaments to Ca2+ in rat ventricular muscle.

Background Bupivacaine exhibits a cardiodepressant effect, the molecular mechanism(s) of which have yet to be fully understood. Bupivacaine may directly act on contractile proteins and thereby decrease myofibrillar Ca2+ sensitivity. Methods Rat ventricular muscle was used. First, the effect of bupivacaine was examined on tetanic contractions in isolated intact myocytes. Next, Triton X-100–treated ventricular trabeculae were used to investigate the effect of bupivacaine on the pCa (= −log [Ca2+])–tension relation as well as on maximal Ca2+-activated tension. Furthermore, to test whether bupivacaine inhibits the pathway downstream from Ca2+ binding to troponin C, tension was elicited in the skinned preparations by lowering the Mg-adenosine triphosphate (MgATP) concentration in the absence of Ca2+. The effect of bupivacaine on the pMgATP (= −log [MgATP])–tension relation was examined. Results In myocytes, 3 &mgr;m bupivacaine significantly (P < 0.01) increased intracellular Ca2+ concentration required for 5% cell shortening from the resting cell length. In skinned preparations, bupivacaine shifted the pCa–tension relation to the lower pCa side; the midpoint of the pCa curve (pCa50) was significantly (P < 0.05) changed by 10 and 100 &mgr;m bupivacaine. A highly correlated linear relation (R = 0.81;P < 0.0005) was present between pCa50 and maximal Ca2+-activated tension. Bupivacaine (10 and 100 &mgr;m) significantly (P < 0.05) shifted the midpoint of the pMgATP–tension relation to the higher pMgATP side. Conclusions Bupivacaine decreases myofibrillar Ca2+ sensitivity in ventricular muscle, and this is coupled with the compounds inhibitory effect on the pathway beyond Ca2+ binding to troponin C, possibly on the actomyosin interaction. The current results may partly explain the overall cardiodepressant effect of bupivacaine in vivo.

Comparative effects of bupivacaine and ropivacaine on intracellular calcium transients and tension in ferret ventricular muscle.

Background:Recent evidence suggests that ropivacaine exerts markedly less cardiotoxicity compared with bupivacaine; however, the mechanisms are not fully understood at the molecular level. Methods:Isolated ferret ventricular papillary muscles were microinjected with the Ca2+-binding photoprotein aequorin, and intracellular Ca2+ transients and tension were simultaneously measured during twitch in the absence and presence of bupivacaine or ropivacaine. Results:Bupivacaine and ropivacaine (10, 30, and 100 &mgr;m) reduced peak systolic [Ca2+]i and tension in a concentration-dependent manner. The effects were significantly greater for bupivacaine, particularly on tension (approximately twofold). The percentage reduction of tension was linearly correlated with that of [Ca2+]i for both anesthetics, with the slope of the relationship being ≈1.0 for ropivacaine and ≈1.3 for bupivacaine (slope difference, P < 0.05), suggesting that the cardiodepressant effect of ropivacaine results predominantly from inhibition of Ca2+ transients, whereas bupivacaine suppresses Ca2+ transients and the reaction beyond Ca2+ transients, i.e., myofibrillar activation, as well. BAY K 8644, a Ca2+ channel opener, abolished the inhibitory effects of ropivacaine on Ca2+ transients and tension, whereas BAY K 8644 only partially inhibited the effects of bupivacaine, particularly the effects on tension. Conclusion:The cardiodepressant effect of bupivacaine is approximately twofold greater than that of ropivacaine. Bupivacaine suppresses Ca2+ transients more markedly than does ropivacaine and reduces myofibrillar activation, which may at least in part underlie the greater inhibitory effect of bupivacaine on cardiac contractions. These results suggest that ropivacaine has a more favorable profile as a local anesthetic in the clinical settings.

Protein kinase A-dependent phosphorylation of ryanodine receptors increases Ca2+ leak in mouse heart.

In heart failure, chronic catecholaminergic stimulation increases diastolic Ca(2+) leak from ryanodine receptors (RyRs) of sarcoplasmic reticulum (SR), possibly due to the phosphorylation of RyRs through the activation of protein kinase A (PKA) or Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In the present study, we attempted to identify which activated kinase is responsible for the enhanced Ca(2+) leak caused by beta-adrenergic stimulation. Trabeculae obtained from the hearts of adult male C57BL/6J mice were treated with isoproterenol and then permeabilized with saponin. To examine SR functions, Ca(2+) in SR was released with caffeine and measured with fluo-3. The Ca(2+) leak in isoproterenol-treated preparations was significantly increased when the PKA-dependent phosphorylation of RyR was increased without the involvement of CaMKII-dependent phosphorylation. Both the increase in Ca(2+) leak and the phosphorylation of RyR were blocked by a PKA inhibitor. Our results show that beta-adrenergic stimulation increases Ca(2+) leak from SR through PKA-dependent phosphorylation of RyR.

Estimation of myofibrillar responsiveness to Ca2+ in isolated rat ventricular myocytes

Abstract To estimate myofibrillar responsiveness to Ca2+, we used the relation between cell length and intracellular [Ca2+] ([Ca2+]i) during tetanic contractions of isolated ventricular myocytes. Enzymatically isolated rat ventricular myocytes were loaded with fura-2 AM (4 µM for 10 min) and excited alternately at 340 nm and 380 nm. The ratio (R) of fura-2 fluorescence at these wavelengths [F(340)/F(380), an index of [Ca2+]i] and cell length (L) were measured simultaneously. Following treatment with thapsigargin (0.2 µM), myocytes were stimulated at 10 Hz for 10 s to produce a tetanic contraction every min and an instantaneous plot of R vs L (R-L trajectory) was constructed. The R-L trajectory followed the same path during cell shortening and re-lengthening, suggesting that dynamic equilibrium between R and L was achieved during tetanus. Increasing the extracellular [Ca2+] from 1 to 8 mM extended the R-L trajectory without a substantial shift of the relation. The Ca2+-sensitizing thiadiazinone derivative, EMD57033 (1 µM), shifted the R-L trajectory to the left (sensitization of the myofibrils to Ca2+), whereas the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-methylxantine (200 µM), shifted the R-L trajectory to the right (desensitization of the myofibrils to Ca2+), in agreement with previous results obtained using skinned preparations. We conclude that the R-L trajectory is useful for estimating the myofibrillar responsiveness to Ca2+ in isolated myocytes and may be beneficial for the evaluation of inotropic agents.

Pulmonary hypertension due to left heart disease causes intrapulmonary venous arterialization in rats

Objective: A rat model of left atrial stenosis–associated pulmonary hypertension due to left heart diseases was prepared to elucidate its mechanism. Methods: Five‐week‐old Sprague–Dawley rats were randomly divided into 2 groups: left atrial stenosis and sham‐operated control. Echocardiography was performed 2, 4, 6, and 10 weeks after surgery, and cardiac catheterization and organ excision were subsequently performed at 10 weeks after surgery. Results: Left ventricular inflow velocity, measured by echocardiography, significantly increased in the left atrial stenosis group compared with that in the sham‐operated control group (2.2 m/s, interquartile range [IQR], 1.9‐2.2 and 1.1 m/s, IQR, 1.1‐1.2, P < .01), and the right ventricular pressure‐to‐left ventricular systolic pressure ratio significantly increased in the left atrial stenosis group compared with the sham‐operated control group (0.52, IQR, 0.54‐0.60 and 0.22, IQR, 0.15‐0.27, P < .01). The right ventricular weight divided by body weight was significantly greater in the left atrial stenosis group than in the sham‐operated control group (0.54 mg/g, IQR, 0.50‐0.59 and 0.39 mg/g, IQR, 0.38‐0.43, P < .01). Histologic examination revealed medial hypertrophy of the pulmonary vein was thickened by 1.6 times in the left atrial stenosis group compared with the sham‐operated control group. DNA microarray analysis and real‐time polymerase chain reaction revealed that transforming growth factor‐&bgr; mRNA was significantly elevated in the left atrial stenosis group. The protein levels of transforming growth factor‐&bgr; and endothelin‐1 were increased in the lung of the left atrial stenosis group by Western blot analyses. Conclusions: We successfully established a novel, feasible rat model of pulmonary hypertension due to left heart diseases by generating left atrial stenosis. Although pulmonary hypertension was moderate, the pulmonary hypertension due to left heart diseases model rats demonstrated characteristic intrapulmonary venous arterialization and should be used to further investigate the mechanism of pulmonary hypertension due to left heart diseases.