Protein tyrosine phosphatase 1B is a mediator of cyclic ADP ribose-induced Ca2+ signaling in ventricular myocytes.

Cyclic ADP-ribose (cADPR) releases Ca2+ from ryanodine receptor (RyR)-sensitive calcium pools in various cell types. In cardiac myocytes, the physiological levels of cADPR transiently increase the amplitude and frequency of Ca2+ (that is, a rapid increase and decrease of calcium within one second) during the cardiac action potential. In this study, we demonstrated that cADPR levels higher than physiological levels induce a slow and gradual increase in the resting intracellular Ca2+ ([Ca2+]i) level over 10 min by inhibiting the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA). Higher cADPR levels mediate the tyrosine-dephosphorylation of α-actin by protein tyrosine phosphatase 1B (PTP1B) present in the endoplasmic reticulum. The tyrosine dephosphorylation of α-actin dissociates phospholamban, the key regulator of SERCA, from α-actin and results in SERCA inhibition. The disruption of the integrity of α-actin by cytochalasin B and the inhibition of α-actin tyrosine dephosphorylation by a PTP1B inhibitor block cADPR-mediated Ca2+ increase. Our results suggest that levels of cADPR that are relatively higher than normal physiological levels modify calcium homeostasis through the dephosphorylation of α-actin by PTB1B and the subsequent inhibition of SERCA in cardiac myocytes.

[Adenoviral short hairpin RNA targeting phosphodiesterase 5 attenuates cardiac remodeling and cardiac dysfunction following myocardial infarction in mice].

OBJECTIVE: To observe the impact of PDE5shRNA on cardiac remodeling and heart function following myocardial infarction in mice. METHODS: Myocardial infarction (MI) was induced in mice by left coronary artery ligation. Mice were randomly assigned to sham group (n = 6), PDE5shRNA group (n = 12), common adenovirus group (n = 15) and DMEM group (n = 8). Four weeks post-MI, the survival rate was evaluated. Cardiac function was examined by echocardiography. HE staining and Masson staining were used to evaluate the myocardial infarction size and fibrosis. The number of blood vessels was evaluated by immunohistochemistry, PDE5 protein expression in the left ventricular was detected using Western blot, level of cGMP or PKG activity in the left ventricle was evaluated with ELISA. RESULTS: Four weeks post-MI, all mice survived in the sham group, 3(37%) mice died in the DMEM group, 1 (8%) died in the PDE5shRNA group and 5 died in the common adenovirus group (33%). Infarct size was significantly reduced in PDE5shRNA group compared with the common adenovirus group and DMEM group [(25.4 ± 2.9)% vs. (42.0 ± 3.2)% and (43.4 ± 2.6) %, P < 0.05]. Cardiac function was significantly improved in PDE5shRNA group compared to common adenovirus group and DMEM group[LVFS: (21.1 ± 3.7)% vs. (14.2 ± 2.9)% and (14.22 ± 2.91)%, all P < 0.05; LVEF: (48.2 ± 7.1)% vs. (34.6 ± 6.2)% and (38.1 ± 2.8)%, all P < 0.05; LVESD: (3.87 ± 0.45) mm vs.(4.91 ± 0.62) mm and (4.63 ± 0.37) mm, all P < 0.05]. The blood vessel density was also higher in PDE5shRNA group compared with common adenovirus group (infarct area:14.3 ± 2.0 vs. 6.6 ± 1.2, P < 0.05; periinfarct area: 23.6 ± 2.1 vs. 13.7 ± 2.4, P < 0.05). Compared with common adenovirus group, level of PDE5 was significantly downregulated and level of cGMP or PKG was significantly upregulated in PDE5shRNA group (all P < 0.05). CONCLUSIONS: Present study suggests PDE5shRNA improves cardiac function and attenuates cardiac remodeling through reducing infarction size and cardiac fibrosis and these beneficial effects are possibly mediated by activating cGMP/PKG signaling pathway.

Beneficial effects of tadalafil on left ventricular dysfunction in doxorubicin-induced cardiomyopathy.

BACKGROUND: It is not clear yet how tadalafil affects nonischemic cardiomyopathy, although its beneficial effects on acute myocardial infarction are well-known. We investigated tadalafil's beneficial effects on nonischemic cardiomyopathy and the specific mechanisms of its effects. METHODS: Cardiomyopathy was induced in mice by a single intraperitoneal injection of doxorubicin (15 mg/kg). In some cases, tadalafil (4 mg/kg/day, p.o., 14 days) was started simultaneously. After two weeks, cardiac function was evaluated by echocardiography and cardiac catheterization, then all of the mice were killed and cardiac specimens were subjected for hemotoxylin and eosin staining, Masson's trichrome staining, terminal deoxynucleotidyltransferase dUTP nick-end labeling assay, enzyme-linked immunosorbent assay, and Western blot. RESULTS: Two weeks later, left ventricular dilatation and dysfunction were apparent in mice given doxorubicin but were significantly attenuated by tadalafil treatment. Tadalafil also protected hearts against doxorubicin-induced cardiomyocyte atrophy/degeneration and myocardial fibrosis. No doxorubicin-induced apoptotic effects were seen between groups. Cardiac cGMP level was lower in the doxorubicin-treated group, however it was significantly increased with tadalafil treatment. Compared to the control group, the myocardial expression of 3 sarcomeric proteins, myosin heavy chain, troponin I, and desmin were significantly decreased in the doxorubicin-treated group, which were restored by the tadalafil treatment. CONCLUSIONS: The present study indicates a protective effect of tadalafil mainly through cGMP signaling pathway against doxorubicin-induced nonischemic cardiomyopathy.

Basic fibroblast growth factor increases intracellular magnesium concentration through the specific signaling pathways.

Basic fibroblast growth factor (bFGF) plays an important role in angiogenesis. However, the underlying mechanisms are not clear. Mg(2+) is the most abundant intracellular divalent cation in the body and plays critical roles in many cell functions. We investigated the effect of bFGF on the intracellular Mg(2+) concentration ([Mg(2+)](i)) in human umbilical vein endothelial cells (HUVECs). bFGF increased [Mg(2+)](i) in a dose-dependent manner, independent of extracellular Mg(2+). This bFGF-induced [Mg(2+)](i) increase was blocked by tyrosine kinase inhibitors (tyrphostin A-23 and genistein), phosphatidylinositol 3-kinase (PI3K) inhibitors (wortmannin and LY294002) and a phospholipase Cgamma (PLCgamma) inhibitor (U73122). In contrast, mitogen-activated protein kinase inhibitors (SB202190 and PD98059) did not affect the bFGF-induced [Mg(2+)](i) increase. These results suggest that bFGF increases the [Mg(2+)](i) from the intracellular Mg(2+) stores through the tyrosine kinase/PI3K/PLCgamma-dependent signaling pathways.

[VEGF165-induced angiogenesis by regulating intracellular free Mg2+ in HUVECs].

AIM: The mechanism of vascular endothelial growth factor165 (VEGF165) on intracellular free magnesium ([Mg2+]i) in human umbilical vein endothelial cells (HUVECs) was investigated. METHODS: [Mg2+]i in HUVECs loaded with fluorescent magnesium indicator mag-fura-2 were quantitatively detected the use of intracellular cation measurement system. RESULTS: VEGF165 significantly increased [Mg2+]i in the extracellular Mg2+ and this effect could be blocked by pretreatment with tyrosine kinase inhibitors (tyrphostin A23 and genistein), phosphatidylinositol 3-kinase (PI3K) inhibitors (wortmannin and LY294002) and phospholipase Cgamma (PLCgamma) inhibitor (U73122). In contrast, phospholipase Cgamma (PLCgamma) inhibitor analog (U73343), mitogen-activated protein kinase inhibitors (SB202190 and PD98059) had no effect on the VEGF165-induced [Mg2+]i increase. CONCLUSION: The increase of [Mg2+]i by VEGF165 originates from intracellular Mg2+ pool through tyrosine kinase/ PI3K/PLCgamma-dependent signaling pathways.

Overexpression of junctate induces cardiac hypertrophy and arrhythmia via altered calcium handling.

Junctate-1 is a newly identified integral endoplasmic/sarcoplasmic reticulum Ca2+ binding protein. However, its functional role in the heart is unknown. In the present study, the consequences of constitutively overexpressed junctate in cardiomyocytes were investigated using transgenic (TG) mice overexpressing junctate-1. TG mice (8 weeks old) showed cardiac remodeling such as marked bi-atrial enlargement with intra-atrial thrombus and biventricular hypertrophy. The TG mice also showed bradycardia with atrial fibrillation, reduced amplitude and elongated decay time of Ca2+ transients, increased L-type Ca2+ current and prolonged action potential durations. Time-course study (2-8 weeks) showed an initially reduced SR function due to down-regulation of SERCA2 and calsequestrin followed by sarcolemmal protein expression and cardiac hypertrophy at later age. These sequential changes could well be correlated with the physiological changes. Adrenergic agonist treatment and subsequent biochemical study showed that junctate-1 TG mice (8 weeks old) were under local PKA signaling that could cause increased L-type Ca2+ current and reduced SR function. Junctate-1 in the heart is closely linked to the homeostasis of E-C coupling proteins and a sustained increase of junctate-1 expression leads to a severe cardiac remodeling and arrhythmias.

[Evidence for a major role of Mg2+ in VEGF165-mediated angiogenesis].

OBJECTIVE: The effect of vascular endothelial growth factor(165) (VEGF(165)) on intracellular free magnesium ([Mg(2+)](i)) and the relationship between Mg(2+) and angiogenesis in human umbilical vein endothelial cells (HUVECs) were investigated in this study. METHODS: [Mg(2+)](i) in HUVECs loaded with fluorescent magnesium indicator mag-fura-2 were quantitatively detected with the use of intracellular cation measurement system. HUVECs were obtained from normal fetus and cultured in M199 with 0.2 fetal bovine serum. The angiogenesis effects of VEGF(165) were observed in presence of 0 mmol/L, 1 mmol/L or 2 mmol/L of extracellular Mg(2+). RESULTS: VEGF(165) significantly increased [Mg(2+)](i) in a dose-dependent manner independent of extracellular Mg(2+), Na(+) and Ca(2+) and this effect could be blocked by pretreatment with VEGF(165) receptor-2 (KDR) inhibitor (SU1498). The angiogenesis induced by VEGF(165) was significantly inhibited cells with 0 mmol/L extracellular Mg(2+), the angiogenesis effects of VEGF(165) were similar in cells with 1 mmol/L and 2 mmol/L extracellular Mg(2+) and these effects could be blocked by SU1498. CONCLUSIONS: These results suggest that the [Mg(2+)](i) increase induced by VEGF(165) originates from intracellular Mg(2+) pools and promotes angiogenesis via KDR-dependent signaling pathways.

Vascular endothelial growth factor increases the intracellular magnesium.

Vascular endothelial growth factor (VEGF) is one of the key players in the process of angiogenesis. However, its underlying mechanism remains unclear. Mg2+ is the most abundant intracellular divalent cation in the body and plays critical roles in many cell functions. We investigated the effect of VEGF on intracellular Mg2+ in human umbilical vein endothelial cells (HUVECs). VEGF-A165 increased the intracellular Mg2+ concentration ([Mg2+]i) in a dose-dependent manner, with or without extracellular Mg2+, and the increase of [Mg2+]i was blocked by pretreatment with SU1498, tyrosine kinase inhibitors (tyrphostin A-23 and genistein), phosphatidylinositol 3-kinase (PI3K) inhibitors (wortmannin and LY294002) or phospholipase Cgamma (PLCgamma) inhibitor (U73122). In contrast, mitogen-activated protein kinase inhibitors (SB202190 and PD98059) had no effect on the VEGF-induced [Mg2+]i increase. These results suggest that VEGF-A165 increases the [Mg2+]i from the intracellular Mg2+ stores through the tyrosine kinase/PI3K/PLCgamma-dependent signaling pathways.