A myosin II ATPase inhibitor reduces force production, glucose transport, and phosphorylation of AMPK and TBC1D1 in electrically stimulated rat skeletal muscle

Abstract

Contraction-stimulated glucose transport by skeletal muscle appears to be caused by the cumulative effects of multiple inputs [potentially including AMP-activated protein kinase (AMPK), Ca(2+) flux, and force production], making it challenging to isolate the roles of these putative regulatory factors. To distinguish the effects of force production from the direct consequences of Ca(2+) flux, the predominantly type II rat epitrochlearis muscle was incubated without (vehicle) or with N-benzyl-p-toluenesulfonamide (BTS), a highly specific myosin II ATPase inhibitor that prevents force production by electrically stimulated (ES) type II fibers without altering cytosolic Ca(2+). In ES muscles, BTS vs. vehicle had an 84% reduction in force production and a 57% decrement in contraction-stimulated 3-O-methylglucose transport (3MGT). BTS did not alter the ES increase in phosphorylation of CaMKII (indicative of cytosolic Ca(2+)) or the amount of glycogen depletion. ES caused significant reductions in ATP (48%) and phosphocreatine (67%) concentrations for vehicle-treated muscles. For BTS-treated muscles, ES did not reduce ATP and caused only a 42% decrease in phosphocreatine. There was an ES increase in phosphorylation of AMPK, acetyl-CoA carboxylase (an AMPK substrate), and TBC1D1 for vehicle-treated muscles but not for BTS-treated muscles. These results point toward an essential role for tension-related events, including AMPK activation, in the 57% contraction-stimulated increase in 3MGT that was inhibited by BTS and further suggest a possible role for TBC1D1 phosphorylation. Non-tension-related events (e.g., increased cytosolic Ca(2+) rather than increased AMPK and TBC1D1 phosphorylation) are implicated in the contraction-stimulated increase in 3MGT that persisted in the presence of BTS.

Fig. 1.

Tension development. Paired epitrochlearis muscles, incubated with 50 μM N-benzyl-p-toluenesulfonamide (BTS) or vehicle, were electrically stimulated to contract (pulse duration of 0.1 ms, pulse rate of 100 pulses/s, train duration of 10 s, train rate of 2 min⁻¹ for 5 min). A: peak developed tension determined for each tetanus was normalized to the muscle wet weight (g). B: area under the force production curve summed for all 10 tetani was calculated by subtracting the baseline tension from total area under the curve. Values are means ± SE (n = 44). *P < 0.0001, BTS vs. vehicle.

Fig. 2.

Rate of 3-O-methylglucose (3-MG) transport with or without contraction and with or without 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR). Paired epitrochlearis muscles were incubated with 50 μM BTS or vehicle. A: muscles were mounted at resting tension (basal) or were electrically stimulated (contraction) before measurement of 3-MG transport. *P < 0.001, basal vs. contraction within the BTS or vehicle treatment groups. †P < 0.001, BTS vs. vehicle with contraction. B: estimated contraction-stimulated increase (estimated-Δ-contraction) was calculated by subtracting the mean value for basal 3-MG transport rate of each treatment from the respective contraction-stimulated value. ‡P < 0.001, estimated-Δ-contraction for BTS vs. vehicle. C: muscles were incubated in the absence (basal) or presence of 2 mM AICAR before 3-MG transport measurement. *P < 0.001, basal vs. AICAR within the BTS or vehicle treatment groups. D: estimated AICAR-stimulated increase (estimated-Δ-AICAR) was calculated by subtracting the mean value for basal 3-MG transport rate of each treatment from the respective AICAR-stimulated value. Values are means ± SE (n = 10–17).

Fig. 3.

Phosphorylation (p) of CaMKII, AMP-activated protein kinase (AMPK), and acetyl-CoA carboxylase (ACC). Paired epitrochlearis muscles were incubated with 50 μM BTS or vehicle. Muscles were mounted at resting tension (basal) or electrically stimulated (contraction) before being freeze-clamped and processed for measurement of pCaMKII (A), pAMPK (B), and pACC (C). A: *P < 0.05, basal vs. contraction within the vehicle treatment group. B and C: *P < 0.001, basal vs. contraction within the vehicle treatment group. †P < 0.01, BTS vs. vehicle with contraction. Values are means ± SE (n = 5–11).

Fig. 4.

Phosphorylation of Ser⁴⁷³Akt, Thr³⁰⁸Akt, and Ser²¹ glycogen synthase kinase-3α (GSK3α Ser²¹). Paired epitrochlearis muscles were incubated with 50 μM BTS or vehicle. Muscles were mounted at resting tension (basal) or electrically stimulated (contraction) before being freeze-clamped and processed for measurement of pSer⁴⁷³Akt (A), pThr³⁰⁸Akt (B), and pGSK3α Ser²¹ (C). A: *P < 0.001, basal vs. contraction within the vehicle treatment group. †P < 0.01, BTS vs. vehicle with contraction. B and C: *P < 0.01, basal vs. contraction within the vehicle and BTS treatment groups. †P < 0.05, BTS vs. vehicle with contraction. Values are means ± SE (n = 9–19).

Fig. 5.

Phosphorylation of Akt substrate of 160 kDa (AS160) and TBC1D1. Paired epitrochlearis muscles were incubated with 50 μM BTS or vehicle. Muscles were mounted at resting tension (basal) or electrically stimulated (contraction) before being freeze-clamped and processed for phosphorylation of AS160 (PAS-AS160; A) or TBC1D1 (PAS-TBC1D1; B). A: for PAS-AS160, samples were immunoprecipitated with anti-PAS and then immunoblotted with anti-AS160. *P < 0.01, basal vs. contraction within the vehicle treatment group. B: for PAS-TBC1D1, samples were immunoprecipitated with anti-TBC1D1 and then immunoblotted with anti-PAS. *P < 0.005, basal vs. contraction within the vehicle treatment group. †P < 0.05, BTS vs. vehicle with contraction. Values are means ± SE (n = 11–14).

Fig. 6.

A model for the effects of myosin II ATPase inhibitor BTS on contraction-stimulated glucose transport (GT) in rat epitrochlearis muscle. This model depicts our interpretation of the results of the current study in the context of previously published research on the mechanisms for contraction-stimulated GT in skeletal muscle and the effects of BTS on skeletal muscle function. BTS selectively inhibits myosin II ATPase, which in turn favors attenuated reduction in ATP concentration and less activation of AMPK in electrically stimulated type II fibers. Because BTS does not inhibit myosin I ATPase, it is not expected to alter ATP concentration or AMPK activation in type I fibers. BTS does not alter Ca²⁺ flux of electrically stimulated muscles, regardless of fiber type. Accordingly, we hypothesize that the 57% reduction in contraction-stimulated GT for BTS- vs. vehicle-treated muscles was attributable to reduced AMPK-associated GT in type II fibers. We further hypothesize that the 43% of contraction-stimulated GT that was not inhibited by BTS was attributable to the summed effects of Ca²⁺-associated GT in type II fibers and GT in type I fibers.