Modification of cardiac disease by transgenically altered histone deacetylase 6.

Histone deacetylase 6 (HDAC6) is known to deacetylate amino acid lysine in alpha-tubulin. However, the functional role of HDAC6 in the progression of cardiac disease remains uncertain. The functional role of HDAC6 in the hearts was examined using transgenic (TG) mice expressing either human wild-type HDAC6, deacetylase inactive HDAC6 (HDAC6H216A, H611A), and human HDAC6 replaced all serine or threonine residues with aspartic acid at N-terminal 1- 43 amino acids (HDAC6NT-allD) specifically in the hearts. Overexpression of wild-type HDAC6 significantly reduced acetylated tubulin levels, and overexpression of HDAC6H216A, H611A significantly increased it in the mouse hearts. Detectable acetylated tubulin disappeared in HDAC6NT-allD TG mouse hearts. Neither histological alteration nor alteration of cardiac function was observed in the HDAC6 TG mouse hearts. To analyze the role of HDAC6 and acetylated tubulin in disease conditions, we examined HDAC6 in isoprenaline-induced hypertrophy or pressure-overload hypertrophy (TAC). No obvious alteration in the heart weight/body weight ratio or gene expressions of hypertrophic markers between NTG and HDAC6NT-allD mice was observed following treatment with isoprenaline. In contrast, a marked reduction in the shortening fraction and dilated chamber dilatation was detected in the HDAC6NT-allD TG mouse hearts 2 weeks after TAC. A sustained low level of acetylated tubulin and acetylated cortactin was observed in the TAC HDAC6NT-allD TG mouse hearts. Cardiac HDAC6 activity that can regulate acetylated levels of tubulin and cortactin may be critical factors involved in cardiac disease such as pressure-overload hypertrophy.

Expression of cell adhesion molecule, Gicerin/CD146 during the formation of heart and in the cardiac hypertrophy.

Gicerin/CD146 is a cell adhesion molecule which belongs to the immunoglobulin (Ig) superfamily. We have reported the existence of gicerin/CD146 in the nervous system, heart, lung and smooth muscles of blood vessels. In this study, we make a cardiac hypertrophy model rat by constricting the rat aorta (AAC, ascending aortic constriction) and examined the effect on the expression of gicerin/CD146 in the heart. We found that the expression level of gicerin/CD146 was increased by the AAC treatment. Next, stretch stimulation was applied to myocardial cell line H9c2 cells to confirm that gicerin/CD146 may participate in the cellular hypertrophy model. We also treated the cells with inhibitors of MAP pathway enzymes. In cultured myocardial cells, the expression level of gicerin/CD146 was increased by the stretch stimulation and decreased by inhibiting the MAP pathway. Based on the above findings, it is suggested that the expression of gicerin/CD146 is involved in cardiac hypertrophy, and that the MAP pathway may be involved in the expression of gicerin/CD146 RNA in the cardiomyocyte. In addition, the expression level of gicerin/CD146 RNA in neonatal rats was upregulated after birth. Therefore, it is suggested that gicerin/CD146 might participate in the increase of myocardial cell volume both in the pathway of cardiac hypertrophy and in the developmental growth of heart.

Chronic HDAC6 Activation Induces Atrial Fibrillation Through Atrial Electrical and Structural Remodeling in Transgenic Mice.

Atrial fibrillation (AF) is a relatively common complication of hypertension. Chronic hypertension induces cardiac HDAC6 catalytic activity. However, whether HDAC6 activation contributes to hypertension-induced AF is still uncertain. We examined whether chronic cardiac HDAC6 activation-induced atrial remodeling, leading to AF induction.The HDAC6 constitutively active transgenic (TG) (HDAC6 active TG) mouse overexpressing the active HDAC6 protein, specifically in cardiomyocytes, was created to examine the effects of chronic HDAC6 activation on atrial electrical and structural remodeling and AF induction in HDAC6 active TG and non-transgenic (NTG) mice. Left atrial burst pacing (S1S1 = 30 msec) for 15-30 sec significantly increased the frequency of sustained AF in HDAC6 active-TG mice compared with NTG mice. Left steady-state atrial pacing (S1S1 = 80 msec) decreased the atrial conduction velocity in isolated HDAC6 active TG compared with NTG mouse atria. The atrial size was similar between HDAC6 active TG and NTG mice. In contrast, atrial interstitial fibrosis increased in HDAC6 active TG compared with that of NTG mouse atria. While protein expression levels of both CX40 and CX43 were similar between HDAC6 active TG and NTG mouse atria, a heterogeneous distribution of CX40 and CX43 occurred in HDAC6 active-TG mouse atria but not in NTG mouse atria. Gene expression of interleukin 6 increased in HDAC6 active TG compared with NTG mouse atria.Chronic cardiac HDAC6 activation induced atrial electrical and structural remodeling, and sustained AF. Hypertension-induced cardiac HDAC6 catalytic activity may play important roles in the development of AF.

Pretreatment with KGA-2727, a selective SGLT1 inhibitor, is protective against myocardial infarction-induced ventricular remodeling and heart failure in mice.

Recent studies demonstrated that sodium-glucose co-transporter 1 (SGLT1) is associated with human ischemic cardiomyopathy. However, whether SGLT1 blockade is effective against ischemic cardiomyopathy is still uncertain. We examined the effects of KGA-2727, a selective SGLT1 inhibitor, on myocardial infarction (MI)-induced ischemic cardiomyopathy. To create MI, left anterior descending coronary artery (LAD) ligation with or without KGA-2727 administration was performed in C57BL/6J mice. Four weeks after the operation, all mice were investigated. Left ventricular fractional shortening (LVFS) was reduced and KGA-2727 significantly improved it in LAD-ligated MI mice. The cardiomyocyte diameter, and ANP, BNP, ß-MHC, and IL-18 gene expressions significantly increased in LAD-ligated mouse left ventricles compared with those of sham-operated mouse left ventricles, and KGA-2727 inhibited increases in them. Myocardial fibrosis and upregulation of CTGF and MMP-3 gene expressions in the left ventricle were increased in LAD-ligated mice compared with sham-operated mice, and KGA-2727 decreased them in the LAD-ligated left ventricles. SGLT1 protein expression level was significantly higher in LAD-ligated compared with sham-operated mouse ventricles regardless of KGA-2727 treatment. These results suggest that KGA-2727 pretreatment protects against MI-induced left ventricular remodeling through SGLT1 blockade and that it may become a new pharmacological therapy for ischemia-induced cardiomyopathy.

8-HEPE-Concentrated Materials from Pacific Krill Improve Plasma Cholesterol Levels and Hepatic Steatosis in High Cholesterol Diet-Fed Low-Density Lipoprotein (LDL) Receptor-Deficient Mice.

Eicosapentaenoic acid (EPA), one of the N-3 polyunsaturated fatty acids (n-3 PUFAs), is a major active ingredient of fish that contributes to improve dyslipidemia. Recently, we demonstrated that 8-hydroxyeicosapentaenoic acid (8-HEPE) had a more positive effect on metabolic syndrome than EPA, and that 8-HEPE induced peroxisome proliferator-activated receptor (PPAR)α activation in the liver. We investigated the effects of 8-HEPE-concentrated materials from Pacific krill on dyslipidemia and hepatic steatosis in low-density lipoprotein (LDL) receptor-deficient (LDLR-KO) mice. Eight-week-old male LDLR-KO mice were fed a Western diet (0.15% cholesterol, WD), WD supplemented with 8-HEPE-concentrated materials from Pacific krill (8-HEPE included; WD +8-HEPE), or a standard diet (SD) for eighteen weeks, respectively. Murine J774.1 macrophages were incubated in the absence or presence of 8-HEPE (50 µM) or EPA (50 µM). 8-HEPE-concentrated materials significantly increased the plasma high-density lipoprotein (HDL)-cholesterol level, and decreased the plasma LDL-cholesterol and hepatic triglyceride levels in WD-fed LDLR-KO mice. Moreover, the rate of Oil Red O-positive staining was higher in the liver of WD-fed LDLR-KO mice than in that of 8-HEPE + WD-fed LDLR-KO mice. 8-HEPE but not EPA significantly increased gene expression levels of ABCA1, CD36, and interleukin 6 (IL-6) in murine J774.1 macrophages compared with those in the control. These results suggest that 8-HEPE-concentrated materials improve dyslipidemia and hepatic steatosis increasing ABCA1, CD36, and IL-6 gene expressions in macrophages.

Cardiac overexpression of perilipin 2 induces atrial steatosis, connexin 43 remodeling, and atrial fibrillation in aged mice.

Atrial fibrillation (AF) is prevalent in patients with obesity and diabetes, and such patients often exhibit cardiac steatosis. Since the role of cardiac steatosis per se in the induction of AF has not been elucidated, the present study was designed to explore the relation between cardiac steatosis and AF. Transgenic (Tg) mice with cardiac-specific overexpression of perilipin 2 (PLIN2) were housed in the laboratory for more than 12 mo before the study. Electron microscopy of the atria of PLIN2-Tg mice showed accumulation of small lipid droplets around mitochondrial chains, and five- to ninefold greater atrial triacylglycerol (TAG) content compared with wild-type (WT) mice. Electrocardiography showed significantly longer RR intervals in PLIN2-Tg mice than in WT mice. Transesophageal electrical burst pacing resulted in significantly higher prevalence of sustained (>5 min) AF (69%) in PLIN2-Tg mice than in WT mice (24%), although it was comparable in younger (4-mo-old) mice. Connexin 43 (Cx43), a gap junction protein, was localized at the intercalated disks in WT atria but was heterogeneously distributed on the lateral side of cardiomyocytes in PLIN2-Tg atria. Langendorff-perfused hearts using the optical mapping technique showed slower and heterogeneous impulse propagation in PLIN2-Tg atria compared with WT atria. Cardiac overexpression of hormone-sensitive lipase in PLIN2-Tg mice resulted in atrial TAG depletion and amelioration of AF susceptibility. The results suggest that PLIN2-induced steatosis is associated with Cx43 remodeling, impaired conduction propagation, and higher incidence of AF in aged mice. Therapies targeting cardiac steatosis could be potentially beneficial against AF in patients with obesity or diabetes.

IL-1ß Plays an Important Role in Pressure Overload-Induced Atrial Fibrillation in Mice.

Hypertension is one risk for atrial fibrillation (AF) and induces cardiac inflammation. Recent evidence indicates that pressure overload-induced ventricular structural remodeling is associated with the activation of nucleotide binding-oligomerization domain (NOD)-like receptor P3 (NLRP3) inflammasomes, including an apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We hypothesized that NLRP3 inflammasomes are an initial sensor for danger signals in pressure overload-induced atrial remodeling, leading to AF. Transverse aortic constriction (TAC) or a sham procedure was performed in mice deficient for ASC-/- and interleukin-1ß (IL-1ß-/-). One week after the procedure, electrical left atrial burst pacing from the esophagus was performed for 30 s to induce AF. IL-1ß, monocyte chemotactic protein 1 (MCP-1), connective tissue growth factor (CTGF), and collagen 1 gene expression were also examined. The electrical burst pacing induced AF in TAC-operated wild-type (WT) (p < 0.001) and ASC-/- (p < 0.05) mice, compared to no AF in the sham-operated WT and ASC-/- mice, respectively. In contrast, the number of mice in which sustained AF was induced was similar between TAC-operated IL-1ß-/- and sham-operated IL-1ß-/- mice (p > 0.05). The expression of all genes tested was increased in TAC-operated WT and ASC-/- mice compared with sham-operated WT and ASC-/- mouse atria, respectively. CTGF and collagen 1, but not MCP-1, gene expressions were increased in TAC-operated IL-1ß-/- mouse atria compared with sham-operated WT and IL-1ß-/- mouse atria. In contrast, the IL-1ß gene was not detected in either TAC-operated or sham-operated IL-1ß-/- mouse atria. These results suggest that an IL-1ß activation pathway, different from NLRP3 inflammasomes, plays an important role in pressure overload-induced sustained AF.

Bcl-2-associated athanogene 3 (BAG3) is an enhancer of small heat shock protein turnover via activation of autophagy in the heart.

Bcl-2-associated athanogene 3 (BAG3) is strongly expressed in both cardiac and skeletal muscle. A recent study showed that BAG3 may play a protective role in muscles. Little is known, however, regarding the detailed role of BAG3 in cardiac muscle. To better understand the functional role of cardiac BAG3 in the heart, we generated transgenic (TG) mice that overexpress BAG3. A decrease in fractional shortening, and the induction of cardiac atrial natriuretic peptide, were observed in BAG3 TG mice. Moreover, a marked reduction in the protein level of small HSPs was detected in BAG3 TG mouse hearts. We analyzed the cardiac small HSP levels when either the ubiquitin-proteasome system (UPS) or the autophagy system (AS) was inhibited in BAG3 TG mice. The protein turnovers of small HSPs by the AS were activated in BAG3 TG mouse hearts. Thus, BAG3 is critical for the protein turnover of small HSPs via activation of autophagy in the heart.

Chronic Pressure Overload Induces Cardiac Hypertrophy and Fibrosis via Increases in SGLT1 and IL-18 Gene Expression in Mice.

Increased gene expression levels of sodium-glucose cotransporter 1 (SGLT1) are associated with hypertrophic and ischemic cardiomyopathy. However, it remains unclear whether chronic pressure overload increases SGLT1 expression, which in turn induces hypertrophic cardiomyopathy. We hypothesized that pressure overload could increase SGLT1 gene expression, leading to the development of hypertrophic cardiomyopathy.To create pressure overload-induced cardiomyopathy, transverse aortic constriction (TAC) was performed in SGLT1-deficient (SGLT1-/-) and wild-type (WT) mice. Six weeks after surgery, all mice were investigated. We observed a reduction of left ventricular fractional shortening and left ventricular dilatation in TAC-operated WT but not in TAC-operated SGLT1-/- mice. SGLT1, interleukin 18, connective tissue growth factor, and collagen type 1 gene expression levels were increased in TAC-operated WT mouse hearts compared with that of sham-operated WT mouse hearts. Moreover, heart/body weight ratio and ventricular interstitial fibrosis were increased in TAC-operated WT mice compared with that of sham-operated WT mice. Interestingly, these factors did not increase in TAC-operated SGLT1-/- mice compared with that of sham-operated WT and SGLT1-/- mice. Phenylephrine, an adrenergic α1 receptor agonist, caused cardiomyocyte hypertrophy in neonatal WT mouse hearts to a significantly larger extent than in neonatal SGLT1-/- mouse hearts.In conclusion, the results indicate that chronic pressure overload increases SGLT1 and IL-18 gene expressions, leading to the development of hypertrophic cardiomyopathy. These results make SGLT1 a potential candidate for the therapeutic target for hypertension-induced cardiomyopathy.

Cardiac overexpression of perilipin 2 induces dynamic steatosis: prevention by hormone-sensitive lipase.

Cardiac intracellular lipid accumulation (steatosis) is a pathophysiological phenomenon observed in starvation and diabetes mellitus. Perilipin 2 (PLIN2) is a lipid droplet (LD)-associated protein expressed in nonadipose tissues, including the heart. To explore the pathophysiological function of myocardial PLIN2, we generated transgenic (Tg) mice by cardiac-specific overexpression of PLIN2. Tg hearts showed accumulation of numerous small LDs associated with mitochondrial chains and high cardiac triacylglycerol (TAG) content [8-fold greater than wild-type (WT) mice]. Despite massive steatosis, cardiac uptake of glucose, fatty acids and VLDL, systolic function, and expression of metabolic genes were comparable in the two genotypes, and no morphological changes were observed by electron microscopy in the Tg hearts. Twenty-four hours of fasting markedly reduced steatosis in Tg hearts, whereas WT mice showed accumulation of LDs. Although activity of adipose triglyceride lipase in heart homogenate was comparable between WT and Tg mice, activity of hormone-sensitive lipase (HSL) was 40-50% less in Tg than WT mice under both feeding and fasting conditions, suggesting interference of PLIN2 with HSL. Mice generated through crossing of PLIN2-Tg mice and HSL-Tg mice showed cardiac-specific HSL overexpression and complete lack of steatosis. The results suggest that cardiac PLIN2 plays an important pathophysiological role in the development of dynamic steatosis and that the latter was prevented by upregulation of intracellular lipases, including HSL.

Transplantation of adipose tissue-derived stem cells improves cardiac contractile function and electrical stability in a rat myocardial infarction model.

The transplantation of adipose tissue-derived stem cells (ADSCs) improves cardiac contractility after myocardial infarction (MI); however, little is known about the electrophysiological consequences of transplantation. The purpose of this study was to clarify whether the transplantation of ADSCs increases or decreases the incidence of ventricular tachyarrhythmias (VT) in a rat model of MI. MI was induced experimentally by permanent occlusion of the left anterior descending artery of Lewis rats. ADSCs were harvested from GFP-transgenic rats, and were cultured until passage four. ADSCs (10×10(6)) resuspended in 100µL saline or pro-survival cocktail (PSC), which enhances cardiac graft survival, were injected directly into syngeneic rat hearts 1week after MI. The recipients of ADSCs suspended in PSC had a larger graft area compared with those receiving ASDCs suspended in saline at 1week post-transplantation (number of graft cells/section: 148.7±10.6 vs. 22.4±3.4, p<0.05, n=5/group). Thereafter, all ADSC recipients were transplanted with ASDCs in PSC. ADSCs were transplanted into infarcted hearts, and the mechanical and electrophysiological functions were assessed. Echocardiography revealed that ADSC recipients had improved contractile function compared with those receiving PSC vehicle (fractional shortening: 21.1±0.9 vs. 14.1±1.2, p<0.05, n≥12/group). Four weeks post-transplantation, VT was induced via in vivo programmed electrical stimulation. The recipients of ADSCs showed a significantly lower incidence of induced VT compared with the control (31.3% vs. 83.3%, p<0.05, n≥12/group). To understand the electrical activity following transplantation, we performed ex vivo optical mapping using a voltage sensitive dye, and found that ADSC transplantation decreased conduction velocity and its dispersion in the peri-infarct area. These results suggest that ADSC transplantation improved cardiac mechanical and electrophysiological functions in subacute MI.

Phlorizin prevents electrically-induced ventricular tachyarrhythmia during ischemia in langendorff-perfused guinea-pig hearts.

Phlorizin is a type of flavonoids and has a peroxynitrite scavenging effect. This study aimed to elucidate the effects of phlorizin on ischemia-induced ventricular tachyarrhythmia (VT). Optical signals from the epicardial surface of the ventricle or left ventricular end diastolic pressure (LVEDP) were recorded during acute global ischemia in 42 Langendorff-perfused guinea pig hearts. Experiments were performed in the control condition and in the presence of phlorizin or N-2-mercaptopropionylglycine (2-MPG), a peroxynitrite scavenger, respectively. Mean action potential duration at 20 min of ischemia did not differ among the three interventions. Impulse conduction time-dependently slowed during 20 min of ischemia in the control. Phlorizin but not 2-MPG improved the ischemic conduction slowing at 15 and 20 min of ischemia. Programmed stimulation induced VT at 20 min of ischemia in the control and in the presence of 2-MPG but not in the presence of phlorizin (p<0.05). LVEDP was increased during 30 min of ischemia in the control and in the presence of 2-MPG but not in the presence of phlorizin. These results indicate that phlorizin prevents VT through the improvement of impulse conduction slowing during ischemia. Phlorizin may be more useful for ischemia-induced VT than 2-MPG.

Nicorandil improves electrical remodelling, leading to the prevention of electrically induced ventricular tachyarrhythmia in a mouse model of desmin-related cardiomyopathy.

1. Transgenic (TG) mice overexpressing an arg120gly missense mutation in heat shock protein B5 (HSPB5; i.e. R120G TG mice) exhibit desmin-related cardiomyopathy. Recently, the cardioprotective effect of nicorandil has been shown to prolong the survival of R120G TG mice. However, whether the TG mice exhibit ventricular arrhythmias and whether nicorandil can inhibit these arrhythmias remain unknown. In the present study we examined the effects of chronic nicorandil administration on ventricular electrical remodelling and arrhythmias in R120G TG mice. 2. Mice were administered nicorandil (15 mg/kg per day) or vehicle (water) orally from 5 to 30 weeks of age. Electrocardiograms (ECG) and optical action potentials were recorded from R120G TG mouse hearts. In addition, the expression of ventricular connexin 43 and the cardiac Na(+) channel Nav1.5 was examined in TG mice. 3. All ECG parameters tested were prolonged in R120G TG compared with non-transgenic (NTG) mice. Nicorandil improved the prolonged P, PQ and QRS intervals in R120G TG mice. Interestingly, impulse conduction slowing and increases in the expression of total and phosphorylated connexin 43 and Nav1.5 were observed in ventricles from R120G TG compared with NTG mice. Nicorandil improved ventricular impulse conduction slowing and normalized the increased protein expression levels of total and phosphorylated connexin 43, but not of Nav1.5, in R120G TG mouse hearts. Electrical rapid pacing at the ventricle induced ventricular tachyarrhythmias (VT) in six of eight R120G TG mouse hearts, but not in any of the eight nicorandil-treated R120G TG mouse hearts (P < 0.05). 4. These findings demonstrate that nicorandil inhibits cardiac electrical remodelling and that the prevention of VT by nicorandil is associated with normalization of connexin 43 expression in this model.

The proximal C-terminus of α(1C) subunits is necessary for junctional membrane targeting of cardiac L-type calcium channels.

In cardiac myocytes, LTCCs (L-type calcium channels) form a functional signalling complex with ryanodine receptors at the JM (junctional membrane). Although the specific localization of LTCCs to the JM is critical for excitation-contraction coupling, their targeting mechanism is unclear. Transient transfection of GFP (green fluorescent protein)-α(1S) or GFP-α(1C), but not P/Q-type calcium channel α(1A), in dysgenic (α(1S)-null) GLT myotubes results in correct targeting of these LTCCs to the JMs and restoration of action-potential-induced Ca2+ transients. To identify the sequences of α(1C) responsible for JM targeting, we generated a range of α(1C)-α(1A) chimaeras, deletion mutants and alanine substitution mutants and studied their targeting properties in GLT myotubes. The results revealed that amino acids L(1681)QAGLRTL(1688) and P(1693)EIRRAIS(1700), predicted to form two adjacent α-helices in the proximal C-terminus, are necessary for the JM targeting of α(1C). The efficiency of restoration of action-potential-induced Ca2+ transients in GLT myotubes was significantly decreased by mutations in the targeting motif. JM targeting was not disrupted by the distal C-terminus of α(1C) which binds to the second α-helix. Therefore we have identified a new structural motif in the C-terminus of α(1C) that mediates the targeting of cardiac LTCCs to JMs independently of the interaction between proximal and distal C-termini of α(1C).

Chronic receptor-mediated activation of Gi/o proteins alters basal t-tubular and sarcolemmal L-type Ca²âº channel activity through phosphatases in heart failure.

L-type Ca(2+) channels (LTCCs) play an essential role in the excitation-contraction coupling of ventricular myocytes. We previously found that t-tubular (TT) LTCC current density was halved by the activation of protein phosphatase (PP)1 and/or PP2A, whereas surface sarcolemmal (SS) LTCC current density was increased by the inhibition of PP1 and/or PP2A activity in failing ventricular myocytes of mice chronically treated with isoproterenol (ISO mice). In the present study, we examined the possible involvement of inhibitory heterotrimeric G proteins (G(i/o)) in these abnormalities by chronically administrating pertussis toxin (PTX) to ISO mice (ISO + PTX mice). Compared with ISO mice, ISO + PTX mice exhibited significantly higher fractional shortening of the left ventricle. The expression level of Gα(i2) proteins was not altered by the treatment of mice with ISO and/or PTX. ISO + PTX myocytes had normal TT and SS LTCC current densities because they had higher and lower availability and/or open probability of TT and SS LTCCs than ISO myocytes, respectively. A selective PKA inhibitor, H-89, did not affect LTCC current densities in ISO + PTX myocytes. A selective PP2A inhibitor, fostriecin, did not affect SS or TT current density in control or ISO + PTX myocytes but significantly increased TT but not SS LTCC current density in ISO myocytes. These results indicate that chronic receptor-mediated activation of G(i/o) in vivo decreases basal TT LTCC activity by activating PP2A and increases basal SS LTCC activity by inhibiting PP1 without modulating PKA in heart failure.

Decrease in the density of t-tubular L-type Ca2+ channel currents in failing ventricular myocytes.

In some forms of cardiac hypertrophy and failure, the gain of Ca(2+)-induced Ca(2+) release [CICR; i.e., the amount of Ca(2+) released from the sarcoplasmic reticulum normalized to Ca(2+) influx through L-type Ca(2+) channels (LTCCs)] decreases despite the normal whole cell LTCC current density, ryanodine receptor number, and sarcoplasmic reticulum Ca(2+) content. This decrease in CICR gain has been proposed to arise from a change in dyad architecture or derangement of the t-tubular (TT) structure. However, the activity of surface sarcolemmal LTCCs has been reported to increase despite the unaltered whole cell LTCC current density in failing human ventricular myocytes, indicating that the "decreased CICR gain" may reflect a decrease in the TT LTCC current density in heart failure. Thus, we analyzed LTCC currents of failing ventricular myocytes of mice chronically treated with isoproterenol (Iso). Although Iso-treated mice exhibited intact t-tubules and normal LTCC subunit expression, acute occlusion of t-tubules of isolated ventricular myocytes with osmotic shock (detubulation) revealed that the TT LTCC current density was halved in Iso-treated versus control myocytes. Pharmacological analysis indicated that kinases other than PKA or Ca(2+)/calmodulin-dependent protein kinase II insufficiently activated, whereas protein phosphatase 1/2A excessively suppressed, TT LTCCs in Iso-treated versus control myocytes. These results indicate that excessive ß-adrenergic stimulation causes the decrease in TT LTCC current density by altering the regulation of TT LTCCs by protein kinases and phosphatases in heart failure. This phenomenon might underlie the decreased CICR gain in heart failure.

Diacylglycerol kinase ζ inhibits ventricular tachyarrhythmias in a mouse model of heart failure.

BACKGROUND: Diacylglycerol kinase ζ (DGKζ) inhibited atrial tachyarrhythmias in a mouse model of heart failure (HF) in our study. However, whether DGKζ prevents the HF-induced ventricular tachyarrhythmia (VT) is unknown. METHODS AND RESULTS: Effects of DGKζ on VT using transgenic mice with transient cardiac expression of activated G protein α(q) (Gα(q)-TG; model of HF) were elucidated and double transgenic mice with cardiac-specific overexpression of both DGKζ and the activated Gα(q) (Gα(q)/DGKζ-TG) were used. Premature ventricular contraction (PVC) and/or VT were frequently observed in Gα(q)-TG mice but not in Gα(q)/DGKζ-TG and wild-type (WT) mice (P<0.01). Protein expressions of canonical transient receptor potential (TRPC) channels 3 and 6 increased in Gα(q)-TG hearts compared with WT and Gα(q)/DGKζ-TG hearts. SK&F96365, a TRPC channel blocker, decreased the number of PVC and prevented VT in anesthetized Gα(q)-TG mice (P<0.05). 1-oleoyl-2-acyl-sn-glycerol (OAG), a diacylglycerol analogue, increased the number of PVC in isolated Gα(q)-TG hearts compared with WT hearts and induced VT in Gα(q)-TG hearts (P<0.01). SK&F96365 decreased the number of PVC and prevented VT in isolated Gα(q)-TG hearts (P<0.01) even in the presence of OAG. Early afterdepolarization (EAD)-induced triggered activity was frequently observed in single Gα(q)-TG ventricular myocytes. Moreover, SK&F96365 prevented the EAD. CONCLUSIONS: These results demonstrated that DGKζ inhibited VT in a mouse model of HF and suggest that TRPC channels participate in VT induction in failing hearts.

Inhibition of untransformed prostaglandin H(2) production and stretch-induced contraction of rabbit pulmonary arteries by indoxam, a selective secretory phospholipase A(2) inhibitor.

Involvement of secretory phospholipase A(2) (sPLA(2)) in the stretch-induced production of untransformed prostaglandin H(2) (PGH(2)) in the endothelium of rabbit pulmonary arteries was investigated. The stretch-induced contraction was significantly inhibited by indoxam, a selective inhibitor for sPLA(2), and NS-398, a selective inhibitor for cyclooxygenase-2 (COX-2). Indoxam inhibited the RGD-sensitive-integrin-independent production of untransformed PGH(2), but did not affect the RGD-sensitive-integrin-dependent production of thromboxane A(2) (TXA(2)). These results suggest that the stretch-induced contraction and untransformed PGH(2) production was mediated by sPLA(2)-COX-2 pathway, making it a new possible target for pharmacological intervention of pulmonary artery contractility.