When ASM cells are exposed to 1 μM ACh, an elevation in [Ca^2 +]_cyt is induced, yet evidence of muscle shortening is delayed by several hundred milliseconds [94]. This time delay reflects the time required for a cascade of events (Fig. 5.2) that can be summarized into four main steps: (1) mobilization of CaM, (2) binding of Ca²⁺ to CaM and subsequent activation of MLCK, (3) phosphorylation of MLC₂₀, and finally (4) crossbridge recruitment. The relative contribution of each the four delays have been quantified in ASM by measuring the response to rapid (virtually instantaneous) release of caged compounds by flash photolysis [94]. The results of this study indicate that mobilization of CaM is the most significant delay, while the delay due to phosphorylation of MLC₂₀ and crossbridge recruitment is minor [94].

The primary role of muscle (skeletal muscle or smooth muscle) is to perform mechanical work through contraction force generation. To perform mechanical work, muscles require chemical energy in the form of ATP hydrolysis. The relationship between muscle work and ATP hydrolysis is not as well defined in smooth muscle compared to skeletal muscle. Therefore, to provide a conceptual framework for the dynamic range of energy demand in smooth muscle, the relationship between mechanical work and energy consumption in skeletal muscle will serve as a point of reference.

The molecular mechanism responsible for contraction in smooth muscle, like all muscle, is the contractile protein apparatus, actin and myosin, conferred by its dual ability to enzymatically harvest chemical energy and, subsequently, perform mechanical work. Chemical energy is harvested via ATP hydrolysis by the actin-activated myosin ATPase (actomyosin ATPase). The energy released by hydrolysis of ATP is coupled to the intrinsic force development by cyclic attachment and detachment of myosin heads to actin (crossbridge cycling).

The intrinsic structural unit that underlies force generation and contraction in skeletal muscle is the actin-myosin crossbridge. Andrew Huxley originally proposed this relationship in a simple model where crossbridge cycling reflected the repetitive transition between two possible mechanical states: (1) myosin attached to actin and generating force and (2) myosin detached from actin [53, 54]. In this model, the hydrolysis of ATP occurs during the transition between attached and detached states. Thus, the Huxley model provides a simplified conceptual framework for both excitation-contraction coupling and excitation-energy coupling.

Skeletal muscle actin-myosin orientation is precisely ordered into units of repeating architecture referred to as sarcomeres – which are important context for application of the Huxley model to skeletal muscle. In brief, the sarcomere is made up of an interlacing, crystalline formation of thick filaments (myosin) and thin filaments (actin). The boundary of a sarcomere is formed by a dense Z-disk or Z-line, which fasten the actin filaments. Actin filaments extend from Z-line to the midline of the sarcomere. The myosin filaments, centered at the midline of the sarcomere, lie between and parallel to actin filaments such that there are six actin filaments surrounding each myosin filament. During muscle contraction, crossbridge cycling within the overlapping regions of the actin and myosin filaments within a half-sarcomere pulls its associated Z-line towards the midline. Thus, the force and contraction vectors are determined by actin-myosin crossbridges within a half-sarcomere. For more details, see review by [93].

The Huxley model has been expanded to a quantitative form by [8, 9]. To summarize, the fraction of strongly bound crossbridges in the force-generating state (Huxley’s two-state model) is represented as “α _fs”. A strongly bound crossbridge is able to supply a unit of force, which is approximated as an averaged quantity of force per crossbridge and designated by “f”. The maximal number of myosin heads available for crossbridges formation within a half-sarcomere is represented as “n”. The combination of these factors determines force (F) generated by a muscle fiber as shown in Eq. 5.1.

The fraction of crossbridges in a force-generating state (α _fs) is a function of the rates of transition between the two mechanical/structural states of the Huxley model as shown in Eq. 5.2 below, where “ƒ_app” is the apparent rate constant for transition to the attached state, and “g _app” is the apparent rate constant for transition to the detached state.

Relationships that characterize skeletal muscle function (force vs. Ca²⁺, force vs. length, and force vs. cross-sectional area) can be accounted for by this model. The parameter α _fs can be increased by an increase in [Ca^2 +]_cyt which promotes transition to the attached, force-generating state of crossbridges (force-Ca²⁺ relationship). Muscle length, which in turn defines sarcomere length and the extent of actin-myosin filament overlap, also affects α _fs without an effect on n (force-length relationship). Finally, fiber cross-sectional area reflects the number of sarcomeres in parallel and, thus, myosin content per half-sarcomere (myosin content determines n) [95].

Using this conceptual framework, a relationship can also be described for ATP consumption assuming a stoichiometry of 1 mole ATP consumed per crossbridge detachment. This relationship is shown in Eq. 5.3 where “b” represents the number of half-sarcomeres within the fiber.

The load opposing the intrinsic crossbridge-induced sarcomeric contraction slows crossbridge cycling rate due to its effect on g _app. From Eq. 5.2, this slowing of g _app has the effect of increasing α _fs and thereby increasing force generation to the point where there is no contraction (isometric force). The rate of contraction (shortening velocity) depends inversely on external load imposed on the sarcomere and therefore determines the force (or load)-velocity relationship. From Eq. 5.3, the rate of ATP hydrolysis (“ATPase” in Eq. 5.3) also depends on g _app; thus, external load and intrinsic force generation opposing the load will influence the rate of ATP hydrolysis. The direct relationship between heat (reflecting energy consumption) and power output of muscle due to contraction (the product of force and shortening velocity) is known as the Fenn effect and was first described in 1923 [29]. Thus, the maximum rate of ATP hydrolysis occurs at peak power output in skeletal muscle [93, 95, 97].

In skeletal muscle fibers, isometric force generation at any given level of Ca^2 + activation is accompanied by a sustained rate of ATP hydrolysis as described by Eq. 5.3 [95] .Thus, the energy cost of force generation does not change during a given level of isometric activation. In contrast, during sustained isometric force generation of smooth muscle, the rate of ATP hydrolysis decreases with time even as force is sustained [25, 48, 104]. This indicates that in smooth muscle, the energy cost of producing force decreases with time – sustained smooth muscle force generation becomes more energy efficient. The energy cost of force generation in smooth muscle can be explained by the Huxley model with consideration of the differences between smooth and skeletal muscle mechanics. For example, there are key differences in actin-myosin alignment, actin-myosin remodeling, and Ca^2 + regulation of crossbridge recruitment. Each of these factors will be considered in the next sections.

Monomeric myosin is assembled into polymers or thick filaments in a nonhelical, side-polar configuration in smooth muscle [50, 110], while in skeletal muscle myosin filaments form a helical, bipolar configuration (Fig. 5.3). The bipolar arrangement forms a “bare area” at the middle of the myosin thick filament. This bare area and the steric constraints of the sarcomeric structure impose limits on muscle length in skeletal muscle that do not exist in smooth muscle. The side-polar arrangement of actin-myosin filaments in smooth muscle allows a significantly greater length range [91, 110].

In both smooth and skeletal muscle, myosin thick filaments interact with actin thin filaments to form crossbridges. However, in skeletal muscle, actin filaments are anchored in a regular fashion by the Z-disks of the sarcomere, whereas in smooth muscle, actin filaments are clustered into regions called dense bodies or membrane-associated dense plaques (Fig. 5.3) [39, 93]. The dense bodies fasten the side-polar actin-myosin lattice and allow force transmission [50, 63] – analogous to the Z-disk in skeletal muscle sarcomeres [39]. However, the internal arrangement of the contractile apparatus (actin-myosin filaments, dense bodies, and dense plaques) in smooth muscle is not regularly ordered and may undergo remodeling during the contractile response (see reviews [41, 91, 113]). Thus, although external isometric conditions may be maintained, dynamic internal remodeling may change the length and overlap relationships between actin and myosin filaments. From Eq. 5.1, force in skeletal muscle is dependent on the number of myosin heads (n) and the fraction of myosin heads forming crossbridges (α _fs). Actin and myosin filament remodeling in smooth muscle will affect both n and α _fs. In addition, actin and myosin filament remodeling will result in changes in internal loading that affect crossbridge cycling rate (g _app from Eq. 5.2), α _fs (Eq. 5.1), and the rate of ATP hydrolysis (Eq. 5.3).

There is evidence of changes in actin polymerization during Ca^2 + activation and force generation in smooth muscle [40, 49, 68]. Actin exists in two distinct but interchangeable forms: polymeric, filamentous actin (F-actin) and monomeric actin (G-actin) [41, 58, 68]. During contraction, there is a decrease in G-actin content in ASM [68]. Pharmacological inhibition of actin polymerization reduces isometric force generation in smooth muscle [40, 58, 68], which suggests that actin polymerization plays an important role in the contractile response. In addition to actin polymerization, there is a reorganization of the contractile network through myosin polymerization [1, 55, 64, 65, 91] that changes the length of the myosin filaments and thus the number of myosin heads that can potentially form crossbridges (n in Eq. 5.1). Filamentous actin is also coupled to the extracellular matrix (ECM) by focal adhesion complexes (membrane-bound dense plaques) [40]. Focal adhesion complexes are comprised of a scaffold of structural and signaling proteins, including mechanosensitive transmembrane integrins [41, 113]. The location of focal adhesion complexes around the PM is dynamically regulated during contraction [3, 32]. In other cell types which are motile, focal adhesions orchestrate actin polymerization to cause cell movement [41, 113]. A similar process may occur in the ASM where focal adhesions recruit actin polymerization (increasing internal loading) according to local mechanical stimuli.

In addition to internal aspects of a contractile stimulus, the ECM transmits mechanical loads (external load) onto the focal adhesions. This external load in vivo is dynamic due to inflation and deflation of the lung [40, 41, 113]. Dynamic changes in external loading may stimulate changes (perhaps via integrin-mediated pathways) in actin-myosin filament architecture. The actin-myosin architecture at any given moment is therefore a function of the history of mechanical stimuli [39, 49, 64] and influences internal loading of crossbridges.

In response to an elevation of [Ca^2 +]_cyt,, crossbridge recruitment in smooth muscle is myosin-activated, whereas in skeletal muscle Ca^2 + regulation of crossbridge recruitment is actin-regulated. Skeletal muscle actin is associated with other proteins, troponin and tropomyosin, that regulate exposure of the myosin-binding site on actin. In skeletal muscle, with an increase in [Ca^2 +]_cyt, troponin C binds Ca^2 +, which induces a conformational change in troponin I and troponin T followed by transposition of tropomyosin uncovering the myosin-binding site on the actin filament. With the myosin-binding site uncovered, crossbridge recruitment and cycling proceeds [93].

In contrast, in smooth muscle, Ca^2 +-dependent regulation of crossbridge recruitment requires phosphorylation of the regulatory domain of myosin (MLC₂₀) by myosin light-chain kinase (MLCK) (Fig. 5.2). MLCK activity is stimulated by an increase in [Ca^2 +]_cyt through the binding of Ca^2 + to calmodulin (CaM). The extent of MLC₂₀ phosphorylation is also regulated by myosin light-chain phosphatase (MLCP), which is not Ca²⁺-dependent but is regulated by a Rho kinase pathway [98]. As a consequence, the phosphorylation of MLC₂₀ reflects the balance between MLCK and MLCP activities and, therefore, Ca^2 +-dependent and independent (i.e., Ca^2 + sensitivity) mechanisms.

Smooth muscle force generation and contraction is generally initiated by a transient elevation of [Ca^2 +]_cyt with an associated transient increase in MLC₂₀ phosphorylation. Yet, force generation in smooth muscle can be maintained at a constant level despite transients in [Ca^2 +]_cyt or MLC₂₀ phosphorylation. Therefore, there are two distinct phases of force generation in smooth muscle initiated by a transient elevation of [Ca^2 +]_cyt: (1) the initial increase in both force and MLC₂₀ phosphorylation and (2) the maintenance of force despite decreasing [Ca^2 +]_cyt and MLC₂₀ phosphorylation. This second phase of smooth muscle activation is also associated with a slowing of shortening velocity (crossbridge cycling rate) and has been termed the “latch state” [44, 69]. According to this hypothesis, a unique crossbridge state – the “latch-bridge” – is formed when the myosin head of an attached crossbridge (due to prior MLC₂₀ phosphorylation) is then dephosphorylated. These “latch-bridges” cycle at a slower rate with an increased duty cycle (amount of time attached vs. detached) and, therefore, maintain force at a lower level of MLC₂₀ phosphorylation or [Ca^2 +]_cyt reflecting increased Ca^2 + sensitivity of force generation. The main assumption of the latch-bridge model is that the extent of MLC₂₀ phosphorylation determines the “latch” or energetic state of smooth muscle force generation [44].

In a Triton X-100 permeabilized ASM preparation [57] directly tested the latch-bridge model by measuring changes in isometric force, MLC₂₀ phosphorylation, and the rate of ATP hydrolysis during maximum Ca²⁺ activation (10 μM Ca²⁺). With maximum Ca²⁺ activation, force increased with a concomitant increase in both MLC₂₀ phosphorylation and the rate of ATP hydrolysis. As Ca²⁺ activation remained constant, isometric force was sustained, as was the level of MLC₂₀ phosphorylation. However, after peaking, the rate of ATP hydrolysis decreased while force and the level of MLC₂₀ phosphorylation were sustained, reflecting a reduction in energy cost, consistent with a “latch” state, but without a change in MLC₂₀ phosphorylation [57]. To further test the latch-bridge hypothesis, thiophosphorylation (accomplished by incubation with ATPγS) was used to prevent dephosphorylation of MLC₂₀ [57]. After thiophosphorylation, the dynamic change in the rate of ATP hydrolysis with sustained Ca^2 + activation was unaffected, also indicating that there is a slowing of crossbridge cycling rate that is independent of any change in the level of MLC₂₀ phosphorylation. This indicates that the level of MLC₂₀ phosphorylation can be dissociated from the rate of ATP hydrolysis and that the “latch-bridge” as proposed does not actually exist.

An alternate “internal loading” hypothesis may explain the decline in the rate of ATP hydrolysis (or energy cost) in ASM during sustained Ca^2 + activation [58]. In this hypothesis, internal loading on crossbridges in ASM is not fixed under isometric conditions, but can change by dynamic cytoskeletal remodeling. It has been demonstrated in ASM that inhibition of actin reorganization with phalloidin (which binds F-actin, preventing its depolymerization) significantly lessens the decline in the rate of ATP hydrolysis during sustained Ca^2 + activation, thereby increasing the energy cost of force generation [58]. The “internal loading” model can be broken down into two phases: (1) an initial phase when crossbridges are recruited by MLC₂₀ phosphorylation (increasing α _fs with an associated increase in the rate of ATP hydrolysis as predicted by Eqs. 5.3 and 5.2) and (2) a delayed phase where there is an increase in internal loading due to cytoskeletal remodeling, with an associated slowing g _app and decrease in the rate of ATP hydrolysis (Eq. 5.3). During the initial phase of Ca²⁺ activation, a portion of the actin-myosin filaments are not fully attached to the internal cytoskeletal structure, resulting in lower internal loading, faster g _app, and a high rate of ATP hydrolysis. Subsequently, with cytoskeletal remodeling, there is an increase in actin-myosin filament attachment to the cytoskeleton imposing increased internal loading with a concomitant slowing of the rate of ATP hydrolysis.

Peak energy consumption of ASM attains a rate of ~0.12 nmol ATP mm⁻³ s⁻¹ at peak power output. This is an approximate threefold increase from the ATP consumption rate during maximum isometric force (~0.04 nmol ATP mm⁻³ s⁻¹) [95]. In contrast, the rate of ATP hydrolysis in skeletal muscle increases from ~1.2 nmol ATP mm⁻³ s⁻¹ during maximum isometric force generation to ~2.7 nmol ATP mm⁻³ s⁻¹ during peak power output [95]. The energy cost of force generation in skeletal muscle ranges from ~16,000–35,000 nmol ATP s⁻¹ mN⁻¹ depending on fiber type. In contrast, the energy cost of force generation in smooth muscle is 6–14-fold lower at ~2,500 nmol ATP s⁻¹ mN⁻¹ [95]. The energy demands of skeletal and smooth muscle are reflections of the quality of their mechanical functions. Smooth muscles perform slower, yet sustained mechanical functions, while skeletal muscles are able to achieve more intense mechanical behaviors – the maximum power output of skeletal muscle is ~10–100 times that produced by smooth muscle [95]. Also, in skeletal muscle, there is a relatively large pool of phosphocreatine (PCr) that is available to buffer ATP hydrolysis transients [77]. In contrast, smooth muscle contains a relatively small PCr pool, which indicates a strong dependence on ATP production rather than on PCr buffering [56, 62, 100]. In summary, it appears that the skeletal muscle contractile response is optimized for maximum power output, while it could be argued that smooth muscle is optimized for maximum energy efficiency. Therefore, efficient matching of energy supply with energy demand may be a more significant governing factor for the ASM contractile response.

The mitochondria provide for most of the energy demands of the cell by replenishing ATP levels through oxidative phosphorylation. The overall outcome of oxidative phosphorylation is to consume both intramitochondrial NADH and O₂ to synthesize ATP from ADP and P_i. In vascular smooth muscle, it is observed that isometric force is strongly correlated to O₂ consumption [56, 62, 77], which implies a dependence on mitochondrial oxidative phosphorylation to supply ATP during contraction. Accordingly, this conceptual framework will define energy homeostasis of the ASM during contraction as a two-part system: ATP hydrolysis by actomyosin ATPase and ATP synthesis by oxidative phosphorylation.