Age-associated differential expression of Na⁺-K⁺-ATPase subunit isoforms in skeletal muscles of F-344/BN rats

Abstract

Skeletal muscle expresses multiple isoforms of the Na(+)-K(+)-ATPase. Their expression has been shown to be differentially regulated under pathophysiological conditions. In addition, previous studies suggest possible age-dependent alterations in Na(+)-K(+) pump function. The present study tests the hypothesis that advancing age is associated with altered Na(+)-K(+)-ATPase enzyme activity and isoform-specific changes in expression of the enzyme subunits. Red and white gastrocnemius (Gast) as well as soleus muscles of male Fischer 344/Brown Norway (F-344/BN) rats at 6, 18, and 30 mo of age were examined. Na(+)-K(+)-ATPase activity, measured by K(+)-stimulated 3-O-methylfluorescein phosphatase activity, increased by approximately 50% in a mixed Gast homogenate from 30-mo-old compared with 6- and 18-mo-old rats. Advancing age was associated with markedly increased alpha(1)- and beta(1)-subunit, and decreased alpha(2)- and beta(2)-subunit in red and white Gast. In soleus, there were similar changes in expression of alpha(1)- and alpha(2)-subunits, but levels of beta(1)-subunit were unchanged. Functional Na(+)-K(+)-ATPase units, measured by [(3)H]ouabain binding, undergo muscle-type specific changes. In red Gast, high-affinity ouabain-binding sites, which are a measure of alpha(2)-isozyme, increased in 30-mo-old rats despite decreased levels of alpha(2)-subunit. In white Gast, by contrast, decreased levels of alpha(2)-subunit were accompanied by decreased high-affinity ouabain-binding sites. Finally, patterns of expression of the four myosin heavy chain (MHC) isoforms (type I, IIA, IIX, and IIB) in these muscles were similar in the three age groups examined. We conclude that, in the skeletal muscles of F-344/BN rats, advancing age is associated with muscle type-specific alterations in Na(+)-K(+)-ATPase activity and patterns of expression of alpha- and beta-subunit isoforms. These changes apparently occurred without obvious shift in muscle fiber types, since expression of MHC isoforms remained unchanged. Some of the alterations occurred between middle-age (18 mo) and senescence (30 mo), and, therefore, may be attributed to aging of skeletal muscle.

Exercise training differentially modifies age-associated alteration in expression of Na+-K+-ATPase subunit isoforms in rat skeletal muscles

“…We demonstrated previously that, in 6-to 30-mo-old Fischer 344 ϫ Brown Norway rats, advancing age is associated with increased levels of ␣ 1 -and ␤ 1 -isoforms and decreased levels of ␣ 2 -and ␤ 2 -isoforms in red and white gastrocnemius (41). Our data demonstrated, for the first time, dynamic regulation of skeletal muscle Na ϩ -K ϩ -ATPase isozymes during aging.…”

“…Total tissue homogenates were prepared as described previously (41). Briefly, skeletal muscles (ϳ200-300 mg) were pulverized and homogenized with a Polytron (Brinkmann Instruments, Westbury, NY) at a speed of 6.5 (11.0 full scale) for three 20-s periods at 4°C in a buffer containing 10 mM Tris ⅐ HCl (pH 7.5), 1 mM EDTA, protease inhibitors (500 M phenylmethylsulfonyl fluoride), 1 M leupeptin, 1 M pepstatin, and 10 M E-64.…”

“…Na ϩ -K ϩ -ATPase subunits were resolved by SDS-PAGE and immunoblotted as previously described (41). For analysis of the ␤-subunits, equal amounts of homogenates (80 g) were first deglycosylated with N-glycosidase F (Glyko, Novato, CA) for 18-20 h at 37°C according to the manufacturer's instructions.…”

Altered expression and insulin-induced trafficking of Na+-K+-ATPase in rat skeletal muscle: effects of high-fat diet and exercise

“…Thus, alterations in the expression of Na ϩ -K ϩ -ATPase iso- forms in skeletal muscle precede the development of insulin resistance on glucose transport. Interestingly, an increase in ␣ 1 -subunit and a decrease in ␣ 2 -subunit expression have been detected in skeletal muscles of aged rats (48), concomitant with an increase in interstitial K ϩ concentrations after contractions (33). Aging is associated with an impaired glucose tolerance, insulin resistance, and diabetes.…”

“…Multiple isoforms of ␣-and ␤-subunits are expressed by and each is encoded by a different gene (34). Adult skeletal muscle expresses two ␣-subunits (␣ 1 and ␣ 2 ) and two ␤-subunits (␤ 1 and ␤ 2 ), and the relative abundance of each isoform and Na ϩ -K ϩ -ATPase activity is regulated in a fiber type-specific manner (26,48,50,51). Following insulin exposure or contraction, Na ϩ -K ϩ -ATPase unit abundance in the plasmalemma and T-tubules of skeletal muscle is increased (4).…”