Efficacy of female rat models in translational cardiovascular aging research.

Cardiovascular disease is the leading cause of death in women in the United States. Aging is a primary risk factor for the development of cardiovascular disease as well as cardiovascular-related morbidity and mortality. Aging is a universal process that all humans undergo; however, research in aging is limited by cost and time constraints. Therefore, most research in aging has been done in primates and rodents; however it is unknown how well the effects of aging in rat models translate into humans. To compound the complication of aging gender has also been indicated as a risk factor for various cardiovascular diseases. This review addresses the systemic pathophysiology of the cardiovascular system associated with aging and gender for aging research with regard to the applicability of rat derived data for translational application to human aging.

Diabetes alters contraction-induced mitogen activated protein kinase activation in the rat soleus and plantaris.

The prescription of anaerobic exercise has recently been advocated for the management of diabetes; however exercise-induced signaling in diabetic muscle remains largely unexplored. Evidence from exercise studies in nondiabetics suggests that the extracellular-signal-regulated kinases (Erk1/2), p38, and c-JUN NH2-terminal kinase (Jnk) mitogen-activated protein kinases (MAPKs) are important regulators of muscle adaptation. Here, we compare the basal and the in situ contraction-induced phosphorylation of Erk1/2- p38- and Jnk-MAPK and their downstream targets (p90rsk and MAPKAP-K2) in the plantaris and soleus muscles of normal and obese (fa/fa) Zucker rats. Compared to lean animals, the time course and magnitude of Erk1/2, p90rsk and p38 phosphorylation to a single bout of contractile stimuli were greater in the plantaris of obese animals. Jnk phosphorylation in response to contractile stimuli was muscle-type dependent with greater increases in the plantaris than the soleus. These results suggest that diabetes alters intramuscular signaling processes in response to a contractile stimulus.

PGF2alpha-associated vascular smooth muscle hypertrophy is ROS dependent and involves the activation of mTOR, p70S6k, and PTEN.

Prostaglandin F2alpha (PGF2alpha) increases reactive oxygen species (ROS) and induces vascular smooth muscle cell (VSMC) hypertrophy by largely unknown mechanism(s). To investigate the signaling events governing PGF2alpha-induced VSMC hypertrophy we examined the ability of the PGF2alpha analog, fluprostenol to elicit phosphorylation of Akt, the mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6k), glycogen synthase kinase-3beta (GSK-3beta), phosphatase and tensin homolog (PTEN), extracellular signal-regulated kinase 1/2 (ERK1/2) and Jun N-terminal kinase (JNK) in growth arrested A7r5 VSMC. Fluprostenol-induced hypertrophy was associated with increased ROS, mTOR translocation from the nucleus to the cytoplasm, along with Akt, mTOR, GSK-3beta, PTEN and ERK1/2 but not JNK phosphorylation. Whereas inhibition of phosphatidylinositol 3-kinase (PI3K) by LY-294002 blocked fluprostenol-induced changes in total protein content, pre-treatment with rapamycin or with the MEK1/2 inhibitor U0126 did not. Taken together, these findings suggest that fluprostenol-induced changes in A7r5 hypertrophy involve mTOR translocation and occur through PI3K-dependent mechanisms.

Load-induced focal adhesion mechanotransduction is altered with aging in the Fischer 344/NNiaHSd x Brown Norway/BiNia rat aorta.

The focal adhesion kinase (FAK) pathway has emerged as a critical component for mediating numerous cellular responses including control of cell growth, differentiation, and adaptation. Here we compared the expression, basal activation, and the ability of increased intraluminal pressure to activate FAK and focal adhesion-associated proteins in the aorta of adult (6 months old) and very aged (36 months old) Fischer 344/NNiaHSd x Brown Norway/BiNia (F344/NXBN) rats. Immunoblot analysis showed increases in the aortic content of FAK (15%), FAK related non-kinase (p41-FRNK) (28%), Src (92%), RhoA (41%), and paxillin (23%) in the very aged aortae. Increased age significantly changed the basal phosphorylation status of FAK and paxillin. Application of aortic intraluminal pressure (200 mm Hg) amplified the phosphorylation of FAK (Tyr 925), Src (Tyr 416), and paxillin (Tyr 188) in adult animals while aortic loading in the very aged animals failed to induce FAK (Tyr 925) phosphorylation. Aging did not alter the load-induced regulation of RhoA; however, FRNK (p41) translocation between cytosolic and membrane compartments was increased. These results confirm previous observations that FAK and focal adhesion-associated proteins are mechanically regulated and expand these studies to suggest that FAK mechanotransduction is altered with aging.

Aging alters mechanical and contractile properties of the Fisher 344/Nnia X Norway/Binia rat aorta.

Vascular mechanical and contractile properties were compared in adult (6 months old) and very-aged (36 months old) Fischer 344/NNiaHSd X Brown Norway/BiNia (F344/NXBN) rats. Our previous work has indicated that aging is associated with aortic medial thickening. This morphological alteration was accompanied by a leftward shift in the aortic stress/strain curve indicating increased vessel stiffness in very-aged animals. Disruption of the endothelium as well as pretreatment of tissues with the nitric oxide (NO) donor sodium nitroprusside eliminated differences, suggesting a link between deficient endothelial NO release and reduced compliance in very-aged aortae. In addition, the Rho kinase inhibitor Y-27632 increased vessel compliance in both adult and very-aged tissues suggesting that the Rho cascade contributed to the stress/strain relationship. Maximal force developed in response to high potassium (K(+)) was reduced by approximately 70% in intact and endothelium-denuded aortae from very-aged rats. In contrast to contractile force development, calcium-dependent stress relaxation was increased in very-aged aorta. Finally, gel electrophoresis indicated a significantly higher tissue content of myosin heavy chain and a higher ratio of SM1/SM2 isoforms with aging. The results suggest multiple molecular changes with aging, which may be expected to alter vascular tissue function.

Sarcopenia-related apoptosis is regulated differently in fast- and slow-twitch muscles of the aging F344/N x BN rat model.

Age-related decreases in muscle mass have been associated with the loss of myonuclei, possibly through a mechanism involving mitochondria. It is unclear if age-related apoptotic mechanisms vary by fiber type. Here we investigate indices of apoptosis along with the regulation of apoptotic mediators in the extensor digitorum longus (EDL) and soleus of adult (6 month), old (30 month), and very old (36 month) Fischer 344/NNiaHSD x Brown Norway/BiNia (F344/N x BN) rats. Compared to 6-month muscles, aged muscles exhibited decreases in muscle mass along with increases in the number of nuclei staining positively for DNA fragmentation. The expression of Bax, Bcl-2, caspase-3 and caspase-9 was regulated differently with aging between muscle types and in a manner not consistent with mitochondria-mediated apoptosis. To investigate the potential of calpain involvement in age-related myonuclear loss, the calpain-dependent cleavage of alpha-fodrin was examined. The proteolytic cleavage of alpha-fodrin by calpains was increased in both muscles with only the 36-month soleus exhibiting increased caspase-dependent alpha-fodrin cleavage. Taken together, these data suggest that apoptotic regulatory events differ between fiber types in the aging F344/N x BN and that mitochondrial-dependent apoptosis pathways may not play a primary role in the loss of muscle nuclei with aging.

Aging influences multiple incidices of oxidative stress in the aortic media of the Fischer 344/NNiaxBrown Norway/BiNia rat.

Here, we determine the influence of aging on multiple markers of oxidative stress in the aorta of adult (6-month), aged (30-month) and very aged (36-month) Fischer 344/NNiaHSdxBrown Norway/BiNia (F344/NxBN) rats. Compared to adults, increases in as determined by oxidation of hydroethidine (HE) to ethidium (Et) were increased 79.7+/-7.0% in 36-month aortae and this finding was highly correlated with increases in medal thickness (r=0.773, p<0.01) and total protein nitration (r=0.706, p<0.01) but not Ki67, a marker for cell proliferation. Regression analysis showed that increases in aortic superoxide anion (O.-2) with aging were significantly correlated with changes in the expression and/or regulation of proteins involved in metabolic (AMPK-alpha), signaling (mitogen activated protein kinases (MAPKs) along with c-Src), apoptotic (Bax, Bcl-2, Traf-2) and transcriptional (NF-kappaB) activities. These results suggest that the aging F344/NxBN aorta may be highly suited for unraveling the molecular events that lead to age-associated alterations in aortic oxidative stress.

Age-associated changes in MAPK activation in fast- and slow-twitch skeletal muscle of the F344/NNiaHSD X Brown Norway/BiNia rat model.

We compared the tissue content, basal phosphorylation, and stretch-induced phosphorylation of the mitogen-activated protein kinase (MAPK) members; extracellular-signal-regulated kinases (ERK 1/2), p38, and c-Jun NH2-terminal kinase (JNK) in the fast-twitch extensor digitorium longus (EDL) and slow-twitch soleus of young adult (6 month), aged (30 month), and very aged (36 month) F344/NNiaHSD X Brown Norway/BiNia (F344/NXBN) rats. The expression and basal phosphorylation of the ERK 1/2, p38, and JNK MAPK proteins were regulated differently with aging in the EDL and soleus. Stretch induced significant phosphorylation of each signaling molecule in both muscle types of young adult and aged animals. In the very aged animals, stretch stimulated ERK 1/2 MAPK phosphorylation; however, EDL stretch failed to induce JNK MAPK phosphorylation, while soleus stretch was unable to induce the phosphorylation of p38 MAPK. The results suggest that skeletal muscle mechanotransduction processes are affected in very aged F344/NXBN rats and that aging alters load-induced signaling in fast- and slow-twitch muscle types differently.

Aging alters vascular mechanotransduction: pressure-induced regulation of p70S6k in the rat aorta.

Physical forces are important regulators of vascular structure and function though it is unknown how aging may affect the ability of the vasculature to respond to mechanical stimuli. We investigated the pressure-induced activation of ribosomal S6-kinase (p70S6k) and its pathway-related proteins (Akt, GSK-3beta, SHP-2, PTEN) in aortae from young adult (6 month), aged (30 month), and very aged (36 month) Fischer 344 x Brown Norway F1 hybrid rats. With aging, the aortic tissue content of Akt. SHP-2, and PTEN was significantly increased while total p70S6k and GSK-3beta were unchanged. By comparison, the basal phosphorylation of p70S6k at Thr 389 and Thr 421/Ser 424 was increased ( approximately 40%) and unchanged, respectively, while Akt decreased (approximately 37%), GSK-3beta was unchanged, SHP-2 increased (approximately 73.5%), and PTEN increased (approximately 120%) in the aortae of very aged rats. Acute pressurization of aortae resulted in similar increases in phosphorylation of Akt among the different age groups. By comparison, pressure-induced phosphorylation of p70S6k at Thr 389, GSK-3beta and SHP-2 decreased; whereas, PTEN dephosphorylation was increased in 36-month versus 6-month aortae. The results indicate marked alterations in the p70S6k signaling pathway with aging. The implications of these findings on age-associated vessel remodeling are discussed.

Regulation of p70S6k, GSK-3beta, and calcineurin in rat striated muscle during aging.

In this study we compared the content and phosphorylation levels of several molecules believed to regulate muscle hypertrophy and fiber type changes in the extensor digitorum longus (EDL), soleus, diaphragm, and heart of adult (6 months), aged (30 months), and very aged (36 months) Fischer 344 x Brown Norway rats. With aging, the mass of the EDL and soleus decreased significantly (approximately 38% and approximately 36%, respectively), the diaphragm's mass remained unchanged while the mass of the heart increased (approximately 35%). Western blotting demonstrated that calcineurin (CnA), the 70-kDa ribosomal S6 kinase (p70(S6k)), glycogen synthase kinase-3beta (GSK-3beta), and the phosphorylated forms of GSK-3beta and p70(S6k) (p-GSK-3beta(Ser9) and p-p70(S6kThr389)) were regulated differently with aging and between muscle types. Total p70(S6k), GSK-3beta, and p-GSK-3beta(Ser9) decreased in the aged-atrophic EDL and soleus while p-p70(S6kThr389) increased. Although total p70(S6k) content diminished in the continuously active diaphragm, phosphorylation of p70(S6k )remained unchanged. Conversely, the expression of GSK-3beta and p-GSK-3beta(Ser9) increased in the diaphragm. With aging, the amount of p-p70(S6kThr389) decreased approximately 56% in the heart while p-GSK-3beta( Ser9) increased approximately 193%. Interestingly, CnA content remained unchanged in the diaphragm, increased approximately 204% in the EDL, and decreased approximately 30% and approximately 65% with aging in the soleus and heart, respectively. These results indicate remarkable differences in the regulation of molecules thought to govern protein synthesis and changes in contractile protein expression.