Determination of rate constants for turnover of myosin isoforms in rat myocardium: implications for in vivo contractile kinetics

Abstract

Locher MR, Razumova MV, Stelzer JE, Norman HS, Patel JR, Moss RL. Determination of rate constants for turnover of myosin isoforms in rat myocardium: implications for in vivo contractile kinetics. Am J Physiol Heart Circ Physiol 297: H247-H256, 2009. First published April 24, 2009 doi:10.1152/ajpheart.00922.2008The ventricles of small mammals express mostly ␣-myosin heavy chain (␣-MHC), a fast isoform, whereas the ventricles of large mammals, including humans, express ϳ10% ␣-MHC on a predominately ␤-MHC (slow isoform) background. In failing human ventricles, the amount of ␣-MHC is dramatically reduced, leading to the hypothesis that even small amounts of ␣-MHC on a predominately ␤-MHC background confer significantly higher rates of force development in healthy ventricles. To test this hypothesis, it is necessary to determine the fundamental rate constants of cross-bridge attachment (fapp) and detachment (gapp) for myosins composed of 100% ␣-MHC or ␤-MHC, which can then be used to calculate twitch time courses for muscles expressing variable ratios of MHC isoforms. In the present study, rat skinned trabeculae expressing either 100% ␣-MHC or 100% ␤-MHC were used to measure ATPase activity, isometric force, and the rate constant of force redevelopment (ktr) in solutions of varying Ca 2ϩ concentrations. The rate of ATP utilization was ϳ2.5-fold higher in preparations expressing 100% ␣-MHC compared with those expressing only ␤-MHC, whereas ktr was 2-fold faster in the ␣-MHC myocardium. From these variables, we calculated fapp to be approximately threefold higher for ␣-MHC than ␤-MHC and gapp to be twofold higher in ␣-MHC. Mathematical modeling of isometric twitches predicted that small increases in ␣-MHC significantly increased the rate of force development. These results suggest that low-level expression of ␣-MHC has significant effects on contraction kinetics.␣-myosin heavy chain; rate constants of cross-bridge attachment and detachment; rate of rise of force THE MYOSIN MOLECULE, a hexamer composed of two heavy chains (ϳ220 kDa) and four light chains (16 -25 kDa), is the principal component of the thick filament and is responsible for the generation of force in mammalian striated muscle. The rod-shaped COOH-terminal domain of the myosin molecule (light meromyosin) forms the backbone of the thick filament, whereas the globular NH 2 -terminus (subfragment 1) contains the actin-binding and catalytic domains (13). Thus, S1 is an important determinant of contractile speed and power developed in the mammalian myocardium.Two isoforms of myosin heavy chain (MHC), ␣ and ␤, have been identified in cardiac muscle (23). These MHC isoforms share 93% amino acid sequence homology (32), and yet they confer distinct functional properties to the myocardium: ␣-MHC has been shown to have markedly higher ATPase activity, faster shortening velocity (V max ), and faster rates of force development (42), whereas ␤-MHC exhibits a lower tension cost and is thus more efficient (1,24,40).Expression levels of the ventricular MHC isoforms are sp...

Altered in vivo left ventricular torsion and principal strains in hypothyroid rats

“…In agreement with previous studies (18,34,50,59), expression of the ␤-MHC isoform in the hypothyroid myocardium slowed k tr by two-to threefold at all levels of activation compared with the euthyroid myocardium (Fig. 3).…”

“…2), confirming that thyroidectomy did not alter the expression and/or phosphorylation status of thin filament proteins (34,59). Changes in titin isoforms have also been reported after long-term thyroidectomy and propylthiouracil treatment (76); however, in this study, we performed in vitro and in vivo mechanical measurements on rats 3 wk after thyroidectomy surgery, where changes in titin isoforms would not be expected.…”

“…expression without altering the expression of other thick and thin filament contractile proteins (18,34,52), such that changes in contractile function between euthyroid and hypothyroid rats can be attributed to the variable expression of MHC isoforms. All mechanical measurements were performed at 22°C, and sarcomere length was set to 2.1 m. The effects of ␣-or ␤-MHC expression on steady-state mechanical properties of the skinned myocardium are shown in Table 1.…”

“…Although the reported decrease in ␣-MHC content in human failing hearts is relatively small (ϳ10% of total MHC), it may have a significant impact on ventricular mechanical function if ␣-MHC is regionally distributed as it is in hearts of other mammalian species (12,62). Several studies (25,26,34,62,67,70) have demonstrated that a relatively small decrease in ␣-MHC expression can have significant impact on in vitro and in vivo contractile performance. Our results cannot directly assess the effects of small decreases of ␣-MHC on global LV mechanical performance because our mechanical measurements were conducted on hypothyroid hearts that lack ventricular ␣-MHC and euthyroid hearts expressing ϳ80% ␣-MHC in the ventricles.…”

Energy cost of isometric force production after active shortening in skinned muscle fibres

“…Metabolic cost was quantified by measuring the ATPase activity using an enzyme-coupled assay (de Tombe and Stienen, 1995;Glyn and Sleep, 1985;Locher et al, 2009;Mateja et al, 2013;Ottenheijm et al, 2011). The activating solution in which the fibres were bathed for 30 s before deactivation after the purely isometric reference contraction and the active shortening contraction was kept and used to quantify the amount of ADP produced (Joumaa and Herzog, 2013).…”