Effects of IGF-1 on I(K) and I(K1) Channels via PI3K/Akt Signaling in Neonatal Cardiac Myocytes.

Previous studies suggest that sarcolemmal potassium currents play important roles in cardiac hypertrophy. IGF-1 contributes to cardiac hypertrophy via activation of PI3K/Akt signaling. However, the relationships between IGF-1, PI3K/Akt signaling and sarcolemmal potassium currents remain unknown. Therefore, we tested the hypothesis that IGF-1 and PI3K/Akt signaling, independently, decrease sarcolemmal potassium currents in cardiac myocytes of neonatal rats. We compared the delayed outward rectifier (I(K)) and the inward rectifier (I(K)) current densities resulting from IGF-1 treatments to those resulting from simulation of PI3K/Akt signaling using adenoviral (Ad) BD110 and wild-type Akt and to those resulting from inhibition of PI3K signaling by LY294002. Ad.BD110 and Ad.Akt decreased I(K) and these decrements were attenuated by LY 294002. The IGF-1 treatments decreased both I(K) and I(K1) but only the I(K) decrement was attenuated by LY294002. These findings demonstrate that IGF-1 may contribute to cardiac hypertrophy by PI3K/Akt signal transduction mechanisms in neonatal rat cardiomyocytes. Failure of LY294002 to effectively antagonize IGF-1 induced decrements in I(K1) suggests that a signal pathway adjunct to PI3K/Akt contributes to IGF-1 protection against arrhythmogenesis in these myocytes. Our findings imply that sarcolemmal outward and inward rectifier potassium channels are substrates for IGF-1/PI3K/Akt signal transduction molecules.

Regulation of the instantaneous inward rectifier and the delayed outward rectifier potassium channels by Captopril and Angiotensin II via the Phosphoinositide-3 kinase pathway in volume-overload-induced hypertrophied cardiac myocytes.

BACKGROUND: Early development of cardiac hypertrophy may be beneficial but sustained hypertrophic activation leads to myocardial dysfunction. Regulation of the repolarizing currents can be modulated by the activation of humoral factors, such as angiotensin II (ANG II) through protein kinases. The aim of this work is to assess the regulation of IK and IK1 by ANG II through the PI3-K pathway in hypertrophied ventricular myocytes. MATERIAL/METHODS: Cardiac eccentric hypertrophy was induced through volume-overload in adult male rats by aorto-caval shunt (3 weeks). After one week half of the rats were given captopril (2 weeks; 0.5 g/l/day) and the other half served as control. The voltage-clamp and western blot techniques were used to measure the delayed outward rectifier potassium current (IK) and the instantaneous inward rectifier potassium current (IK1) and Akt activity, respectively. RESULTS: Hypertrophied cardiomyocytes showed reduction in IK and IK1. Treatment with captopril alleviated this difference seen between sham and shunt cardiomyocytes. Acute administration of ANG II (10-6M) to cardiocytes treated with captopril reduced IK and IK1 in shunts, but not in sham. Captopril treatment reversed ANG II effects on IK and IK1 in a PI3-K-independent manner. However in the absence of angiotensin converting enzyme inhibition, ANG II increased both IK and IK1 in a PI3-K-dependent manner in hypertrophied cardiomyocytes. CONCLUSIONS: Thus, captopril treatment reveals a negative effect of ANG II on IK and IK1, which is PI3-K independent, whereas in the absence of angiotensin converting enzyme inhibition IK and IK1 regulation is dependent upon PI3-K.

Regulation of L-type inward calcium channel activity by captopril and angiotensin II via the phosphatidyl inositol 3-kinase pathway in cardiomyocytes from volume-overload hypertrophied rat hearts.

Heart failure can be caused by pro-hypertrophic humoral factors such as angiotensin II (Ang II), which regulates protein kinase activities. The intermingled responses of these kinases lead to the early compensated cardiac hypertrophy, but later to the uncompensated phase of heart failure. We have shown that although beneficial, cardiac hypertrophy is associated with modifications in ion channels that are mainly mediated through mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (PI3K) activation. This study evaluates the control of L-type Ca(2+) current (I(Ca,L)) by the Ang II/PI3K pathway in hypertrophied ventricular myocytes from volume-overload rats using the perforated patch-clamp technique. To assess activation of the I(Ca,L) in cardiomyocytes, voltages of 350 ms in 10 mV increments from a holding potential of -85 mV were applied to cardiocytes, with a pre-pulse to -45 mV for 300 ms. Volume overload-induced hypertrophy reduces I(Ca,L), whereas addition of Ang II alleviates the hypertrophic-induced decrease in a PI3K-dependent manner. Acute administration of Ang II (10(-6) mol/L) to normal adult cardiomyocytes had no effect; however, captopril reduced their basal I(Ca,L). In parallel, captopril regressed the hypertrophy and inverted the Ang II effect on I(Ca,L) seemingly through a PI3K upstream effector. Thus, it seems that regression of cardiac hypertrophy by captopril improved I(Ca,L) partly through PI3K.

Cross-talk between MAPKs and PI-3K pathways alters the functional density of I(K) channels in hypertrophied hearts.

Mitogen activated protein kinases (MAPK), such as ERK1/2 and p38 MAPK and phosphatidylinositol-3 phosphate kinase (PI-3K) play a major role in the development of cardiac hypertrophy. Recently, we have shown their crucial role in the regulation of the myocardial function through their effects on crucial ion channels. It is the focus of this study to resolve the interaction between these pathways and its implication on the function of the normal and hypertrophied cardiomyocytes. To that end, we created arteriovenous fistula in the adult rat that developed volume-overload eccentric cardiac hypertrophy over a 3-week period. We measured the relative activity of ERK1/2, p38 MAPKs and Akt through western blot analysis and assessed the functional density of the outward rectifier potassium current (I(K)) using the patch-clamp technique. The results showed a mutual negative autoregulation between ERK1/2 and p38 in normal cardiomyocytes, which disappears during cardiac hypertrophy. In addition, PI-3K seems to assume a greater role in mediating IGF-1 effects on the MAPKs during cardiac hypertrophy. This was also relevant to I(K) functional density which was reduced by activation of both MAPKs and Akt by angiotensin II (ANG II) and insulin-like growth factor-1 (IGF-1), respectively; however, this reduction was reversed by inhibition of PI-3K alone in hypertrophied myocytes but not in normal ones. This raises an important implication relative to the role of IGF-1-dependent activation of PI-3K, which may translate into a differential prognostic for cardiac hypertrophy among ethnic groups. This is true in African Americans, having higher circulating IGF-1 levels, and especially true for the athletes among them.

Basal and IGF-I-dependent regulation of potassium channels by MAP kinases and PI3-kinase during eccentric cardiac hypertrophy.

The potassium channels I(K) and I(K1), responsible for the action potential repolarization and resting potential respectively, are altered during cardiac hypertrophy. The activation of insulin-like growth factor-I (IGF-I) during hypertrophy may affect channel activity. The aim was to examine the modulatory effects of IGF-I on I(K) and I(K1) through mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways during hypertrophy. With the use of specific inhibitors for ERK1/2 (PD98059), p38 MAPK (SB203580) and PI3K/Akt (LY294002), Western blot and whole cell patch-clamp were conducted on sham and aorto-caval shunt-induced hypertrophy adult rat myocytes. Basal activation levels of MAPKs and Akt were increased during hypertrophy. Acute IGF-I (10(-8) M) enhanced basal activation levels of these kinases in normal hearts but only those of Akt in hypertrophied ones. I(K) and I(K1) activities were lowered by IGF-I. Inhibition of ERK1/2, p38 MAPK, or Akt reduced basal I(K) activity by 70, 32, or 50%, respectively, in normal cardiomyocytes vs. 53, 34, or 52% in hypertrophied ones. However, basal activity of I(K1) was reduced by 45, 48, or 45% in the former vs. 63, 43, or 24% in the latter. The inhibition of either MAPKs or Akt alleviated IGF-I effects on I(K) and I(K1). We conclude that basal I(K) and I(K1) are positively maintained by steady-state Akt and ERK activities. K+ channels seem to be regulated in a dichotomic manner by acutely stimulated MAPKs and Akt. Eccentric cardiac hypertrophy may be associated with a change in the regulation of the steady-state basal activities of K+ channels towards MAPKs, while that of the acute IGF-I-stimulated ones toward Akt.

Blockade of MyD88 attenuates cardiac hypertrophy and decreases cardiac myocyte apoptosis in pressure overload-induced cardiac hypertrophy in vivo.

In this study, we evaluated whether blocking myeloid differentiation factor-88 (MyD88) could decrease cardiac myocyte apoptosis following pressure overload. Adenovirus expressing dominant negative MyD88 (Ad5-dnMyD88) or Ad5-green fluorescent protein (GFP) (Ad5-GFP) was transfected into rat hearts (n = 8/group) immediately followed by aortic banding for 3 wk. One group of rats (n = 8) was subjected to aortic banding for 3 wk without transfection. Sham surgical operation (n = 8) served as control. The ratios of heart weight to body weight (HW/BW) and heart weight to tibia length (HW/TL) were calculated. Cardiomyocyte size was examined by FITC-labeled wheat germ agglutinin staining of membranes. Cardiac myocyte apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and myocardial interstitial fibrosis was examined by Masson's Trichrome staining. Aortic banding significantly increased the HW/BW by 41.0% (0.44 +/- 0.013 vs. 0.31 +/- 0.008), HW/TL by 47.2% (42.7 +/- 1.30 vs. 29.0 +/- 0.69), cardiac myocyte size by 49.6%, and cardiac myocyte apoptosis by 11.5%, and myocardial fibrosis and decreased cardiac function compared with sham controls. Transfection of Ad5-dnMyD88 significantly reduced the HW/BW by 18.2% (0.36 +/- 0.006 vs. 0.44 +/- 0.013) and HW/TL by 22.3% (33.2 +/- 0.95 vs. 42.7 +/- 1.30) and decreased cardiomyocyte size by 56.8%, cardiac myocyte apoptosis by 76.2%, as well as fibrosis, and improved cardiac function compared with aortic-banded group. Our results suggest that MyD88 is an important component in the Toll-like receptor-4-mediated nuclear factor-kappaB activation pathway that contributes to the development of cardiac hypertrophy. Blockade of MyD88 significantly reduced cardiac hypertrophy, cardiac myocyte apoptosis, and improved cardiac function in vivo.

E-cadherin polymorphisms and haplotypes influence risk for prostate cancer.

BACKGROUND: The E-cadherin (CDH1) gene has been implicated in prostate cancer (PCA) risk, however, the exact mechanism is unknown. Several polymorphisms, such as the C/A variant -160 base pairs from the transcription start site, in the CDH1 gene promoter region have been associated with cancer risk, mainly in European descent populations. METHODS: We screened the entire coding region and 3.0 kilobases of the CDH1 promoter for polymorphisms in 48 African Americans using dHPLC. Twenty-one (21) polymorphisms were observed. Four polymorphisms, including -160C/A, were genotyped in a genetic association study using incident PCA cases (N = 427) and unaffected controls (N = 337) of similar age from three different ethnic groups consisting of African Americans, Jamaicans, and European Americans. RESULTS: We observed a significantly higher frequency of the -160A allele among European American PCA patients (27.5%) compared to the control group (19.7%) (P = 0.04). More importantly, among men of European ancestry under the age of 65 who possess the -160 A allele there was over three times increased risk for prostate cancer (P = 0.05). Also, the AACT haplotype bearing the -160A allele was significantly associated with PCA in European Americans (P = 0.04). CONCLUSIONS: Our data indicate that CDH1 likely is a low-penetrant PCA susceptibility gene, however, population differences in linkage disequilibrium within the CDH1 gene region may influence the effect of susceptibility alleles such as -160A.

Attenuation of cardiac hypertrophy by inhibiting both mTOR and NFkappaB activation in vivo.

A role for the PI3K/Akt/mTOR pathway in cardiac hypertrophy has been well documented. We reported that NFkappaB activation is needed for cardiac hypertrophy in vivo. To investigate whether both NFkappaB activation and PI3K/Akt/mTOR signaling participate in the development of cardiac hypertrophy, two models of cardiac hypertrophy, namely, induction in caAkt-transgenic mice and by aortic banding in mice, were employed. Rapamycin (2 mg/kg/daily), an inhibitor of the mammalian target of rapamycin, and the antioxidant pyrrolidine dithiocarbamate (PDTC; 120 mg/kg/daily), which can inhibit NFkappaB activation, were administered to caAkt mice at 8 weeks of age for 2 weeks. Both rapamycin and PDTC were also administered to the mice immediately after aortic banding for 2 weeks. Administration of either rapamycin or PDTC separately or together to caAkt mice reduced the ratio of heart weight/body weight by 21.54, 32.68, and 42.07% compared with untreated caAkt mice. PDTC administration significantly reduced cardiac NFkappaB activation by 46.67% and rapamycin significantly decreased the levels of p70S6K by 34.20% compared with untreated caAkt mice. Similar results were observed in aortic-banding-induced cardiac hypertrophy in mice. Our results suggest that both NFkappaB activation and the PI3K/Akt signaling pathway participate in the development of cardiac hypertrophy in vivo.

Reduced cardiac hypertrophy in toll-like receptor 4-deficient mice following pressure overload.

OBJECTIVE: We have previously demonstrated that nuclear factor kappa B (NFkappaB) activation is needed for the development of cardiac hypertrophy in vivo. NFkappaB is a downstream transcription factor in the Toll-like receptor (TLR)-mediated signaling pathway; therefore, we investigated a role of TLR4 in cardiac hypertrophy in vivo. METHODS: TLR4-deficient mice (C.C3H-Tlr4(lps-d), n = 6), wild-type (WT) genetic background mice (BALB/c, n = 6), TLR4-deleted strain (C57BL/10ScCr, n = 8), and WT controls (C57BL/10ScSn, n = 8) were subjected to aortic banding for 2 weeks. Age-matched surgically operated mice served as controls. In a separate experiment, rapamycin (2 mg/kg, daily) was administered to TLR4-deficient mice and WT mice immediately following aortic banding. The ratio of heart weight/body weight (HW/BW) was calculated, and cardiac myocyte size was examined by FITC-labeled wheat germ agglutinin staining of membranes. NFkappaB binding activity and the levels of phospho-p70S6K in the myocardium were also examined. RESULTS: Aortic banding significantly increased the ratio of HW/BW by 33.9% (0.601 +/- 0.026 vs. 0.449 +/- 0.004) and cell size by 68.4% in WT mice and by 10.00% (0.543 +/- 0.011 vs. 0.495 +/- 0.005) and by 11.8% in TLR4-deficient mice, respectively, compared with respective sham controls. NFkappaB binding activity and phospho-p70S6K levels were increased by 182.6% and 115.2% in aortic-banded WT mice and by 78.0% and 162.0% in aortic-banded TLR4-deficient mice compared with respective sham controls. In rapamycin-treated aortic-banded mice, the ratio of HW/BW was increased by 18.0% in WT mice and by 3.5% in TLR4-deficient mice compared with respective sham controls. CONCLUSION: Our results demonstrate that TLR4 is a novel receptor contributing to the development of cardiac hypertrophy in vivo and that both the TLR4-mediated pathway and PI3K/Akt/mTOR signaling are involved in the development of cardiac hypertrophy in vivo.

Regulation of atrial contraction by PKA and PKC during development and regression of eccentric cardiac hypertrophy.

ANG II plays a major role in development of cardiac hypertrophy through its AT1 receptor subtype, whereas angiotensin-converting enzyme (ACE) inhibitors are effective in reversing effects of ANG II on the heart. The objective of this study was to investigate the role of PKA and PKC in the contractile response of atrial tissue during development and ACE inhibitor-induced regression of eccentric hypertrophy induced by aortocaval shunt. At 1 wk after surgery, sham and shunt rats were divided into captopril-treated and untreated groups for 2 wk. Then isometric contraction was assessed by electrical stimulation of isolated rat left atrial preparations superfused with Tyrode solution in the presence or absence of specific inhibitors KT-5720 (for PKA) and Ro-32-0432 (for PKC) and high Ca2+. Peak tension developed was greater in shunt than in sham hearts. However, when expressed relative to tissue mass, hypertrophied muscle showed weaker contraction than muscle from sham rats. In sham rats, peak tension developed was more affected by PKC than by PKA inhibition, whereas this differential effect was reduced in the hypertrophied heart. Treatment of shunt rats with captopril regressed left atrial hypertrophy by 67% and restored PKC-PKA differential responsiveness toward sham levels. In the hypertrophied left atria, there was an increase in the velocity of contraction and relaxation that was not evident when expressed in specific relative terms. Treatment with ACE inhibitor increased the specific velocity of contraction, as well as its PKC sensitivity, in shunt rats. We conclude that ACE inhibition during eccentric cardiac hypertrophy produces a negative trophic and a positive inotropic effect, mainly through a PKC-dependent mechanism.