Ozgur Ogut

Regulation of force in vascular smooth muscle

Vascular smooth muscle contraction plays a defining role in the regulation and maintenance of blood pressure, and its deregulation is associated with many clinical syndromes including hypertension, coronary vasospasm and congestive heart failure. Over the past 20 years, there has been a growing understanding of the regulation of 20 kDa myosin light chain phosphorylation by myosin light chain kinase and myosin light chain phosphatase, the role of splice-variant isoforms of both the myosin heavy chain and the essential myosin light chain, as well as the signaling pathways involved in smooth muscle contraction under normal and pathophysiological conditions. This review will attempt to recapitulate the data in the field, primarily focusing on the contractile response of smooth muscle, and the molecular determinants responsible for the regulation of vascular tone.

Acidic and basic troponin T isoforms in mature fast-twitch skeletal muscle and effect on contractility

Developmentally regulated alternative RNA splicing generates distinct classes of acidic and basic troponin T (TnT) isoforms. In fast-twitch skeletal muscles, an acidic-to-basic TnT isoform switch ensures basic isoform expression in the adult. As an exception, an acidic segment in the NH2-terminal variable region of adult chicken breast muscle TnT isoforms is responsible for the unique exclusive expression of acidic TnTs in this muscle (O. Ogut and J.-P. Jin. J. Biol. Chem. 273: 27858-27866, 1998). To understand the relationship between acidic vs. basic TnT isoform expression and muscle contraction, the contractile properties of fibers from adult chicken breast muscle were compared with those of the levator coccygeus muscle, which expresses solely basic TnT isoforms. With use of Triton X-100-skinned muscle fibers, the force and stiffness responses to Ca2+ were measured. Relative to the levator coccygeus muscle, the breast muscle fibers showed significantly increased sensitivity to Ca2+ of force and stiffness with a shift of approximately 0.15 in the pCa at which force or stiffness was 50% of maximal. The expression of tropomyosin, troponin I, and troponin C isoforms was also determined to delineate their contribution to thin-filament regulation. The data indicate that TnT isoforms differing in their NH2-terminal charge are able to alter the sensitivity of the myofibrillar contractile apparatus to Ca2+. These results provide evidence linking the regulated expression of distinct acidic and basic TnT isoform classes to the contractility of striated muscle.Developmentally regulated alternative RNA splicing generates distinct classes of acidic and basic troponin T (TnT) isoforms. In fast-twitch skeletal muscles, an acidic-to-basic TnT isoform switch ensures basic isoform expression in the adult. As an exception, an acidic segment in the NH2-terminal variable region of adult chicken breast muscle TnT isoforms is responsible for the unique exclusive expression of acidic TnTs in this muscle (O. Ogut and J.-P. Jin. J. Biol. Chem. 273: 27858-27866, 1998). To understand the relationship between acidic vs. basic TnT isoform expression and muscle contraction, the contractile properties of fibers from adult chicken breast muscle were compared with those of the levator coccygeus muscle, which expresses solely basic TnT isoforms. With use of Triton X-100-skinned muscle fibers, the force and stiffness responses to Ca2+ were measured. Relative to the levator coccygeus muscle, the breast muscle fibers showed significantly increased sensitivity to Ca2+ of force and stiffness with a shift of ∼0.15 in the pCa at which force or stiffness was 50% of maximal. The expression of tropomyosin, troponin I, and troponin C isoforms was also determined to delineate their contribution to thin-filament regulation. The data indicate that TnT isoforms differing in their NH2-terminal charge are able to alter the sensitivity of the myofibrillar contractile apparatus to Ca2+. These results provide evidence linking the regulated expression of distinct acidic and basic TnT isoform classes to the contractility of striated muscle.

Nonmuscle myosin is regulated during smooth muscle contraction

The participation of nonmuscle myosin in force maintenance is controversial. Furthermore, its regulation is difficult to examine in a cellular context, as the light chains of smooth muscle and nonmuscle myosin comigrate under native and denaturing electrophoresis techniques. Therefore, the regulatory light chains of smooth muscle myosin (SM-RLC) and nonmuscle myosin (NM-RLC) were purified, and these proteins were resolved by isoelectric focusing. Using this method, intact mouse aortic smooth muscle homogenates demonstrated four distinct RLC isoelectric variants. These spots were identified as phosphorylated NM-RLC (most acidic), nonphosphorylated NM-RLC, phosphorylated SM-RLC, and nonphosphorylated SM-RLC (most basic). During smooth muscle activation, NM-RLC phosphorylation increased. During depolarization, the increase in NM-RLC phosphorylation was unaffected by inhibition of either Rho kinase or PKC. However, inhibition of Rho kinase blocked the angiotensin II-induced increase in NM-RLC phosphorylation. Additionally, force for angiotensin II stimulation of aortic smooth muscle from heterozygous nonmuscle myosin IIB knockout mice was significantly less than that of wild-type littermates, suggesting that, in smooth muscle, activation of nonmuscle myosin is important for force maintenance. The data also demonstrate that, in smooth muscle, the activation of nonmuscle myosin is regulated by Ca(2+)-calmodulin-activated myosin light chain kinase during depolarization and a Rho kinase-dependent pathway during agonist stimulation.

Developmentally Regulated, Alternative RNA Splicing-generated Pectoral Muscle-specific Troponin T Isoforms and Role of the NH2-terminal Hypervariable Region in the Tolerance to Acidosis

The structure-function relationship of the alternative RNA splicing-generated NH2-terminal variable region of troponin T (TnT) is essential for understanding the physiological significance of developmental or muscle-specific TnT isoforms. Representing the hypervariable nature of the NH2-terminal region, a repeating transition metal-binding sequence (H(E/A)EAH)4–7 (Tx) has been found in chicken fast skeletal muscle TnT. In the present study, the developmentally regulated pectoral muscle-specific expression of this novel TnT isoform has been characterized. It was found that the variable amino terminus determined the isoelectric points of the TnT isoforms expressed, and the adult muscle-specific inclusion of the Tx sequence resulted in pectoralis TnTs, which were significantly more acidic in their NH2-terminal segment versus gastrocnemius TnTs. Experiments testing the effect of pH on TnT interaction with troponin I and tropomyosin indicated that although the interaction of acidic TnT isoforms with troponin I was less sensitive to the decrease of pH than the basic TnTs, the binding affinity of acidic TnT isoforms with tropomyosin was minimally affected by the decreased pH in contrast to basic TnT isoforms. Given that the majority of adult skeletal muscles express basic fast TnT isoforms, the switching between acidic and basic TnT isoforms may play a role in the functional adaptation of muscle to acidosis.

Interactions between Nebulin-like Motifs and Thin Filament Regulatory Proteins

Nebulin (600–900 kDa) and nebulette (107–109 kDa) are two homologous thin filament-associated proteins in skeletal and cardiac muscles, respectively. Both proteins are capped with a unique region at the amino terminus as well as a serine-rich linker domain and SH3 domains at the COOH terminus. Their significant size difference is attributed to the length of the central region wherein both proteins are primarily composed of ∼35 amino acid repeats termed nebulin-like repeats or motifs. These motifs are marked by a conserved SXXXY sequence and high affinity binding to F-actin. To further characterize the effects that nebulin-like proteins may have on the striated muscle thin filament, we have cloned, expressed, and purified a five-motif chicken nebulette fragment and tested its interaction with the thin filament regulatory proteins. Both tropomyosin and troponin T individually bound the nebulette fragment, although the affinity of this interaction was significantly increased when tropomyosin-troponin T was tested as a binary complex. The addition of troponin I to the tropomyosin-troponin T complex decreased the binding to the nebulette fragment, indicating an involvement of the conserved T2 region of troponin T in this interaction. F-actin cosedimentation demonstrated that the nebulette fragment was able to significantly increase the affinity of the tropomyosin-troponin assembly for F-actin. The relationships provide a means for nebulin-like motifs to participate in the allosteric regulation of striated muscle contraction.

MYPT1 Protein Isoforms Are Differentially Phosphorylated by Protein Kinase G

Background: The mechanism by which PKGIα activates MLC phosphatase with an LZ+ (but not LZ−) MYPT1 isoform is unknown. Results: LZ+ MYPT1 fragments are rapidly phosphorylated by PKGIα at Ser-667 and Ser-694. Conclusion: MYPT1 isoform expression is important for determining the response of vascular beds to NO and NO-based vasodilators. Significance: MYPT1 isoform expression plays a central role in the regulation of vascular tone in health and disease. Smooth muscle relaxation in response to NO signaling is due, in part, to a Ca2+-independent activation of myosin light chain (MLC) phosphatase by protein kinase G Iα (PKGIα). MLC phosphatase is a trimeric complex of a 20-kDa subunit, a 38-kDa catalytic subunit, and a 110–133-kDa myosin-targeting subunit (MYPT1). Alternative mRNA splicing produces four MYPT1 isoforms, differing by the presence or absence of a central insert and leucine zipper (LZ). The LZ domain of MYPT1 has been shown to be important for PKGIα-mediated activation of MLC phosphatase activity, and changes in LZ+ MYPT1 isoform expression result in changes in the sensitivity of smooth muscle to NO-mediated relaxation. Furthermore, PKGIα has been demonstrated to phosphorylate Ser-694 of MYPT1, but phosphorylation at this site does not always accompany cGMP-mediated smooth muscle relaxation. This study was designed to determine whether MYPT1 isoforms are differentially phosphorylated by PKGIα. The results demonstrate that purified LZ+ MYPT1 fragments are rapidly phosphorylated by PKGIα at Ser-667 and Ser-694, whereas fragments lacking the LZ domain are poor PKGIα substrates. Mutation of Ser-667 and Ser-694 to Ala and/or Asp showed that Ser-667 phosphorylation is more rapid than Ser-694 phosphorylation, suggesting that Ser-667 may play an important role in the activation of MLC phosphatase. These results demonstrate that MYPT1 isoform expression is important for determining the heterogeneous response of vascular beds to NO and NO-based vasodilators, thereby playing a central role in the regulation of vascular tone in health and disease.

Impact of actin glutathionylation on the actomyosin-S1 ATPase.

Glutathionylation of intracellular proteins is an established physiological regulator of protein function. In multiple models, including ischemia-reperfusion of the heart, increased oxidative stress results in the glutathionylation of sarcomeric actin. We hypothesized that actin glutathionylation may play a role in the multifactorial change in cardiac muscle contractility observed during this pathophysiological state. Therefore, the functional impact of glutathionylated actin on the interaction with myosin-S1 was examined. Substituting glutathionylated F-actin for unmodified F-actin reduced the maximum actomyosin-S1 ATPase, and this was accompanied by an increase in the activation energy of the steady state ATPase. Measurement of steady state binding did not suggest a large impact of actin glutathionylation on the binding to myosin-S1. However, transient binding and dissociation kinetics determined by stopped-flow methods demonstrated that although actin glutathionylation did not significantly alter the rate constant of myosin-S1 binding, there was a significant decrease in the rate of ATP-induced myosin-S1 detachment in the presence of ADP. These results suggest that actin glutathionylation may play a limited but defined role in the alteration of contractility following oxidative stress to the myocardium, particularly through a decrease in the actomyosin ATPase activity.

Creatine Phosphate Consumption and the Actomyosin Crossbridge Cycle in Cardiac Muscles

Abstract— To investigate the regulation of the actomyosin crossbridge cycle in cardiac muscles, the effects of ATP, ADP, Pi, and creatine phosphate (CP) on the rate of force redevelopment (ktr) were measured. We report that CP is a primary determinant in controlling the actomyosin crossbridge cycling kinetics of cardiac muscles, because a reduction of CP from 25 to 2.5 mmol/L decreased ktr by 51% despite the presence of 5 mmol/L MgATP. The effects of CP on ktr were not a reflection of reduced ATP or accumulated ADP, because lowering ATP to 1 mmol/L or increasing ADP to 1 mmol/L did not significantly decrease ktr. Therefore, the effect of CP on the actomyosin crossbridge cycle is proposed to occur through a functional link between ADP release from myosin and its rephosphorylation by CP–creatine kinase to regenerate ATP. In activated fibers, the functional link influenced the kinetics of activated crossbridges without affecting the aggregate number of force-generating crossbridges. This was demonstrated by the ability of CP to affect ktr in maximally and submaximally activated fibers without altering the force per cross-sectional area. The data also confirm the important contribution of strong binding crossbridges to cardiac muscle activation, likely mediated by cooperative recruitment of adjacent crossbridges to maximize force redevelopment against external load. These data provide additional insight into the role of CP during pathophysiological conditions such as ischemia, suggesting that decreased CP may serve as a primary determinant in the observed decline of dP/dt.

Sildenafil and B-Type Natriuretic Peptide Acutely Phosphorylate Titin and Improve Diastolic Distensibility In VivoClinical Perspective

Background— In vitro studies suggest that phosphorylation of titin reduces myocyte/myofiber stiffness. Titin can be phosphorylated by cGMP-activated protein kinase. Intracellular cGMP production is stimulated by B-type natriuretic peptide (BNP) and degraded by phosphodiesterases, including phosphodiesterase-5A. We hypothesized that a phosphodiesterase-5A inhibitor (sildenafil) alone or in combination with BNP would increase left ventricular diastolic distensibility by phosphorylating titin. Methods and Results— Eight elderly dogs with experimental hypertension and 4 young normal dogs underwent measurement of the end-diastolic pressure-volume relationship during caval occlusion at baseline, after sildenafil, and BNP infusion. To assess diastolic distensibility independently of load/extrinsic forces, the end-diastolic volume at a common end-diastolic pressure on the sequential end-diastolic pressure-volume relationships was measured (left ventricular capacitance). In a separate group of dogs (n=7 old hypertensive and 7 young normal), serial full-thickness left ventricular biopsies were harvested from the beating heart during identical infusions to measure myofilament protein phosphorylation. Plasma cGMP increased with sildenafil and further with BNP (7.31±2.37 to 26.9±10.3 to 70.3±8.1 pmol/mL; P<0.001). Left ventricular diastolic capacitance increased with sildenafil and further with BNP (51.4±16.9 to 53.7±16.8 to 60.0±19.4 mL; P<0.001). Changes were similar in old hypertensive and young normal dogs. There were no effects on phosphorylation of troponin I, troponin T, phospholamban, or myosin light chain-1 or -2. Titin phosphorylation increased with sildenafil and BNP, whereas titin-based cardiomyocyte stiffness decreased. Conclusion— Short-term cGMP-enhancing treatment with sildenafil and BNP improves left ventricular diastolic distensibility in vivo, in part by phosphorylating titin.

Sildenafil and BNP Acutely Phosphorylate Titin and Improve Diastolic Distensibility in Vivo

Background— In vitro studies suggest that phosphorylation of titin reduces myocyte/myofiber stiffness. Titin can be phosphorylated by cGMP-activated protein kinase. Intracellular cGMP production is stimulated by B-type natriuretic peptide (BNP) and degraded by phosphodiesterases, including phosphodiesterase-5A. We hypothesized that a phosphodiesterase-5A inhibitor (sildenafil) alone or in combination with BNP would increase left ventricular diastolic distensibility by phosphorylating titin. Methods and Results— Eight elderly dogs with experimental hypertension and 4 young normal dogs underwent measurement of the end-diastolic pressure-volume relationship during caval occlusion at baseline, after sildenafil, and BNP infusion. To assess diastolic distensibility independently of load/extrinsic forces, the end-diastolic volume at a common end-diastolic pressure on the sequential end-diastolic pressure-volume relationships was measured (left ventricular capacitance). In a separate group of dogs (n=7 old hypertensive and 7 young normal), serial full-thickness left ventricular biopsies were harvested from the beating heart during identical infusions to measure myofilament protein phosphorylation. Plasma cGMP increased with sildenafil and further with BNP (7.31±2.37 to 26.9±10.3 to 70.3±8.1 pmol/mL; P<0.001). Left ventricular diastolic capacitance increased with sildenafil and further with BNP (51.4±16.9 to 53.7±16.8 to 60.0±19.4 mL; P<0.001). Changes were similar in old hypertensive and young normal dogs. There were no effects on phosphorylation of troponin I, troponin T, phospholamban, or myosin light chain-1 or -2. Titin phosphorylation increased with sildenafil and BNP, whereas titin-based cardiomyocyte stiffness decreased. Conclusion— Short-term cGMP-enhancing treatment with sildenafil and BNP improves left ventricular diastolic distensibility in vivo, in part by phosphorylating titin.