Myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC-2) also known as the regulatory light chain of myosin (RLC) is a protein that in humans is encoded by the MYL2 gene. This cardiac ventricular RLC isoform is distinct from that expressed in skeletal muscle (MYLPF), smooth muscle (MYL12B) and cardiac atrial muscle (MYL7).

Ventricular myosin light chain-2 (MLC-2v) refers to the ventricular cardiac muscle form of myosin light chain 2 (Myl2). MLC-2v is a 19-KDa protein composed of 166 amino acids, that belongs to the EF-hand Ca²⁺ binding superfamily. MLC-2v interacts with the neck/tail region of the muscle thick filament protein myosin to regulate myosin motility and function.

Cardiac, ventricular RLC is an 18.8 kDa protein composed of 166 amino acids. RLC and the second ventricular light chain, essential light chain (ELC, MYL3), are non-covalently bound to IQXXXRGXXXR motifs in the 9 nm S1-S2 lever arm of the myosin head, both alpha (MYH6) and beta (MYH7) isoforms. Both light chains are members of the EF-hand superfamily of proteins, which possess two helix-loop-helix motifs in two globular domains connected by an alpha-helical linker.

The N-terminal EF-hand domain of RLC binds calcium/magnesium at activating concentrations, however the dissociation rate is too slow to modulate cardiac contractility on a beat-by-beat basis. Perturbing the calcium binding region of RLC through site-directed mutagenesis (D47A) decreased tension and stiffness in isolated, skinned skeletal muscle fibers, suggesting that the conformational change induced by calcium binding to RLC is functionally important.

Another mode of RLC modulation lies in its ability to be modified by phosphorylation and deamidation in the N-terminal region, resulting in significant charge alterations of the protein. RLC is phosphorylated by a cardiac-specific myosin light chain kinase (MYLK3), which was recently cloned. Studies have supported a role for myosin phosphatase targeting subunit 2 (MYPT2, PPP1R12B) in the dephosphorylation of RLC. Human RLC has an Asparagine at position 14 (Threonine in mouse) and a Serine at position 15 (same in mouse). Endogenous RLC exists as a mixture of unmodified (typically ~50%), singly-modified (either N14 deamidation or S15 phosphorylation) and doubly modified (N14 deamidation and S15 phosphorylation) protein. Both deamidation and phosphorylation contribute negative charge to the N-terminal region of RLC, undoubtedly altering its interaction with the C-terminal myosin alpha helical domain. Functional studies have supported a role for RLC phosphorylation in modulating cardiac myosin crossbridge kinetics. It is well established that RLC phosphorylation enhances myofilament sensitivity to calcium in isometrically-contracting, skinned cardiac fibers. It was also demonstrated that a lack of RLC phosphorylation decreases tension cost (isometric force/ATPase rate at a given pCa), suggesting that RLC phosphorylation augments cycling kinetics of myosin. It has been proposed that RLC phosphorylation promotes a "swing-out" of myosin heads, facilitating weak-to-strong crossbridge binding to actin per unit calcium. Additional insights regarding RLC phosphorylation in beating hearts have come from in vivo studies. Adult mice expressing a non-phosphorylatable cardiac RLC (TG-RLC(P-)) exhibited significant decreases in load-dependent and load-independent measures of contractility. In TG-RLC(P-), the time for the heart to reach peak elastance during ejection was elongated, ejection capacity was decreased and the inotropic response to dobutamine was blunted. It is also clear that ablation of RLC phosphorylation in vivo induces alterations in the phosphorylation of other sarcomeric proteins, namely cardiac myosin binding protein C and cardiac troponin I. Moreover, RLC phosphorylation, specifically, appears to be necessary for a normal inotropic response to dobutamine. In agreement with these findings, a second in vivo model, cardiac myosin light chain kinase (MYLK3) knockout (cMLCK neo/neo), showed depressed fractional shortening, progressing to left ventricular hypertrophy by 4–5 months of age. Taken together, these studies clearly demonstrate that RLC phosphorylation regulates cardiac dynamics in beating hearts, and is critical for eliciting a normal sympathetic response.

MLC-2v plays an essential role in early embryonic cardiac development and function. and represents one of the earliest markers of ventricular specification. During early development (E7.5-8.0), MLC-2v is expressed within the cardiac crescent. The expression pattern of MLC-2v becomes restricted to the ventricular segment of the linear heart tube at E8.0 and remains restricted within the ventricle into adulthood.

Recent studies have highlighted a critical role for MLC2v phosphorylation in cardiac torsion, function and disease. In cardiac muscle, the critical phosphorylation sites have been identified as Ser14/Ser15 in the mouse heart and Ser15 in the human heart. The major kinase responsible for MLC-2v phosphorylation has been identified as cardiac myosin light chain kinase (MLCK), encoded for by Mylk3. Loss of cardiac MLCK in mice results in loss of cardiac MLC-2v phosphorylation and cardiac abnormalities.

Mutations in MYL2 have been associated with familial hypertrophic cardiomyopathy (FHC). Ten FHC mutations have been identified in RLC: E22K, A13T, N47K, P95A, F18L, R58Q, IVS6-1G>C, L103E, IVS5-2A>G, D166V. The first three-E22K, A13T and N47K-have been associated with an unusual mid-ventricular chamber obstruction type of hypertrophy. Three mutations-R58Q, D166V and IVS5-2-are associated with more malignant outcomes, manifesting with sudden cardiac death or at earlier ages. Functional studies demonstrate that FHC mutations in RLC affect its ability to both be phosphorylated and to bind calcium/magnesium.