Rapamycin treatment augments both protein ubiquitination and Akt activation in pressure-overloaded rat myocardium.

Ubiquitin-mediated protein degradation is necessary for both increased ventricular mass and survival signaling for compensated hypertrophy in pressure-overloaded (PO) myocardium. Another molecular keystone involved in the hypertrophic growth process is the mammalian target of rapamycin (mTOR), which forms two distinct functional complexes: mTORC1 that activates p70S6 kinase-1 to enhance protein synthesis and mTORC2 that activates Akt to promote cell survival. Independent studies in animal models show that rapamycin treatment that alters mTOR complexes also reduces hypertrophic growth and increases lifespan by an unknown mechanism. We tested whether the ubiquitin-mediated regulation of growth and survival in hypertrophic myocardium is linked to the mTOR pathway. For in vivo studies, right ventricle PO in rats was conducted by pulmonary artery banding; the normally loaded left ventricle served as an internal control. Rapamycin (0.75 mg/kg per day) or vehicle alone was administered intraperitoneally for 3 days or 2 wk. Immunoblot and immunofluorescence imaging showed that the level of ubiquitylated proteins in cardiomyocytes that increased following 48 h of PO was enhanced by rapamycin. Rapamycin pretreatment also significantly increased PO-induced Akt phosphorylation at S473, a finding confirmed in cardiomyocytes in vitro to be downstream of mTORC2. Analysis of prosurvival signaling in vivo showed that rapamycin increased PO-induced degradation of phosphorylated inhibitor of κB, enhanced expression of cellular inhibitor of apoptosis protein 1, and decreased active caspase-3. Long-term rapamycin treatment in 2-wk PO myocardium blunted hypertrophy, improved contractile function, and reduced caspase-3 and calpain activation. These data indicate potential cardioprotective benefits of rapamycin in PO hypertrophy.

Dasatinib Attenuates Pressure Overload Induced Cardiac Fibrosis in a Murine Transverse Aortic Constriction Model.

Reactive cardiac fibrosis resulting from chronic pressure overload (PO) compromises ventricular function and contributes to congestive heart failure. We explored whether nonreceptor tyrosine kinases (NTKs) play a key role in fibrosis by activating cardiac fibroblasts (CFb), and could potentially serve as a target to reduce PO-induced cardiac fibrosis. Our studies were carried out in PO mouse myocardium induced by transverse aortic constriction (TAC). Administration of a tyrosine kinase inhibitor, dasatinib, via an intraperitoneally implanted mini-osmotic pump at 0.44 mg/kg/day reduced PO-induced accumulation of extracellular matrix (ECM) proteins and improved left ventricular geometry and function. Furthermore, dasatinib treatment inhibited NTK activation (primarily Pyk2 and Fak) and reduced the level of FSP1 positive cells in the PO myocardium. In vitro studies using cultured mouse CFb showed that dasatinib treatment at 50 nM reduced: (i) extracellular accumulation of both collagen and fibronectin, (ii) both basal and PDGF-stimulated activation of Pyk2, (iii) nuclear accumulation of Ki67, SKP2 and histone-H2B and (iv) PDGF-stimulated CFb proliferation and migration. However, dasatinib did not affect cardiomyocyte morphologies in either the ventricular tissue after in vivo administration or in isolated cells after in vitro treatment. Mass spectrometric quantification of dasatinib in cultured cells indicated that the uptake of dasatinib by CFb was greater that that taken up by cardiomyocytes. Dasatinib treatment primarily suppressed PDGF but not insulin-stimulated signaling (Erk versus Akt activation) in both CFb and cardiomyocytes. These data indicate that dasatinib treatment at lower doses than that used in chemotherapy has the capacity to reduce hypertrophy-associated fibrosis and improve ventricular function.

ß3 integrin in cardiac fibroblast is critical for extracellular matrix accumulation during pressure overload hypertrophy in mouse.

The adhesion receptor ß3 integrin regulates diverse cellular functions in various tissues. As ß3 integrin has been implicated in extracellular matrix (ECM) remodeling, we sought to explore the role of ß3 integrin in cardiac fibrosis by using wild type (WT) and ß3 integrin null (ß3-/-) mice for in vivo pressure overload (PO) and in vitro primary cardiac fibroblast phenotypic studies. Compared to WT mice, ß3-/- mice upon pressure overload hypertrophy for 4 wk by transverse aortic constriction (TAC) showed a substantially reduced accumulation of interstitial fibronectin and collagen. Moreover, pressure overloaded LV from ß3-/- mice exhibited reduced levels of both fibroblast proliferation and fibroblast-specific protein-1 (FSP1) expression in early time points of PO. To test if the observed impairment of ECM accumulation in ß3-/- mice was due to compromised cardiac fibroblast function, we analyzed primary cardiac fibroblasts from WT and ß3-/- mice for adhesion to ECM proteins, cell spreading, proliferation, and migration in response to platelet derived growth factor-BB (PDGF, a growth factor known to promote fibrosis) stimulation. Our results showed that ß3-/- cardiac fibroblasts exhibited a significant reduction in cell-matrix adhesion, cell spreading, proliferation and migration. In addition, the activation of PDGF receptor associated tyrosine kinase and non-receptor tyrosine kinase Pyk2, upon PDGF stimulation were impaired in ß3-/- cells. Adenoviral expression of a dominant negative form of Pyk2 (Y402F) resulted in reduced accumulation of fibronectin. These results indicate that ß3 integrin-mediated Pyk2 signaling in cardiac fibroblasts plays a critical role in PO-induced cardiac fibrosis.

Vitamin D status as related to race and feeding type in preterm infants.

BACKGROUND: Despite the higher prevalence of vitamin D deficiency in blacks, the vitamin D status of black preterm infants remains unknown. In addition, with the combination of parenteral and enteral nutritional support that preterm infants receive, the effect of vitamin D-deficient breast milk on vitamin D status is unknown. OBJECTIVE: To evaluate vitamin D status of preterm infants through the first month after delivery and compare status by race and feeding type. STUDY DESIGN: Thirty-six (36) preterm (< or =32 weeks gestation) infants (19 black, 17 white) had assessment of feeding type, vitamin D intake, and serum 25-hydroxyvitamin D [25(OH)D] as a marker of vitamin D status at three time points in the first month after delivery. RESULTS: Black infants had a significantly lower mean 25(OH)D level on day 7-8 and day 14-15 evaluations than white infants [14.9 +/- 6.6 versus 23.3 +/- 9.3 ng/mL (p = 0.021) and 18.3 +/- 7.3 versus 25.6 +/- 10.3 ng/mL (p = 0.048), respectively], but the difference was no longer significant by day 28-30 evaluation [19.6 +/- 7.7 versus 26.2 +/- 11.6 ng/mL (p = 0.26)]. Vitamin D status was not significantly lower in infants receiving predominantly breast milk (p = 0.6). Vitamin D intake rose through the month as the amount and caloric density of enteral nutrition increased. Six infants had significant decrease in serum 25(OH)D values from day 14-15 to day 28-30 evaluation despite receiving > 400 IU/day vitamin D. CONCLUSION: Differences in vitamin D status occurred between black and white infants and were significant through the first 2 weeks after delivery. Infants receiving predominantly breast milk did not have significantly worse vitamin D status than those receiving formula. The significant decline in serum 25(OH)D status observed in 28% of the infants was not related to breast milk intake.