Images in cardiovascular medicine. Case of anomalous right superior vena cava.
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Adult↗
Biomedical subjects
Frank V Brozovich
Publications and source records attributed to Frank V Brozovich.
PHI-1 interacts with the catalytic subunit of myosin light chain phosphatase to produce a Ca(2+) independent increase in MLC(20) phosphorylation and force in avian smooth muscle.
In avian smooth muscles, GTPgammaS produces a Rho kinase mediated increase in PHI-1 phosphorylation and force, but whether this correlation is causal is unknown. We examined the effect of phosphorylated PHI-1 (P-PHI-1) on force and myosin light chain (MLC(20)) phosphorylation at a constant [Ca(2+)]. P-PHI-1, but not PHI-1, increased MLC(20) phosphorylation and force, and phosphorylation of PHI-1 increased the interaction of PHI-1 with PP1c. Microcystin induced a dose-dependent reduction in the binding of PHI-1 to PP1c. These results suggest PHI-1 inhibits myosin light chain phosphatase by interacting with the active site of PP1c to produce a Ca(2+) independent increase in MLC(20) phosphorylation and force.
Animals↗
Effects of h1-calponin ablation on the contractile properties of bladder versus vascular smooth muscle in mice lacking SM-B myosin.
The functional significance of smooth muscle-specific h1-calponin up-regulation in the smooth muscle contractility of SM-B null mice was studied by generating double knockout mice lacking both h1-calponin and SM-B myosin. The double knockout mice appear healthy, reproduce well and do not show any smooth muscle pathology. Loss of h1-calponin in the SM-B null mice bladder resulted in increased maximal shortening velocity (V(max)) and steady-state force generation. The force dilatation pressure, which was decreased in the SM-B null mesenteric vessels, was restored to wild-type levels in the double knockout vessels. In contrast, the half-time to maximal constriction was significantly increased in the double knockout vessels similar to that of SM-B null mice and indicating decreased shortening velocity in the double knockout vessels. Biochemical analyses showed that there is a significant reduction in smooth muscle alpha-actin levels, whereas h-caldesmon levels are increased in the double knockout bladder and mesenteric vessels, suggesting that these changes may also partly contribute to the altered contractile function. Taken together, our studies suggest that up-regulation of h1-calponin in the SM-B null mice may be necessary to maintain a reduced level of cross-bridge cycling over time in the absence of SM-B myosin and play an important role in regulating the smooth muscle contraction.
Actins↗
MYPT1 mutants demonstrate the importance of aa 888-928 for the interaction with PKGIalpha.
During nitric oxide signaling, type Ialpha cGMP-dependent protein kinase (PKGIalpha) activates myosin light chain (MLC) phosphatase through an interaction with the 130-kDa myosin targeting subunit (MYPT1), leading to dephosphorylation of 20-kDa MLC and vasodilatation. It has been suggested that the MYPT1-PKGIalpha interaction is mediated by the COOH-terminal leucine zipper (LZ) of MYPT1 and the NH(2)-terminal LZ of PKGIalpha (HK Surks and ME Mendelsohn. Cell Signal 15: 937-944, 2003; HK Surks et al. Science 286: 1583-1587, 1999), but we previously showed that PKGIalpha interacts with LZ-positive (LZ+) and LZ-negative (LZ-) MYPT1 isoforms (13). Interestingly, PKGIalpha is known to preferentially bind to RR and RK motifs (WR Dostmann et al. Proc Natl Acad Sci USA 97: 14772-14777, 2000), and there is an RK motif within the aa 888-928 sequence of MYPT1 in LZ+ and LZ- isoforms. Thus, to localize the domain of MYPT1 important for the MYPT1-PKGIalpha interaction, we designed four MYPT1 fragments that contained both the aa 888-928 sequence and the downstream LZ domain (MYPT1FL), lacked both the aa 888-928 sequence and the LZ domain (MYPT1TR), lacked only the aa 888-928 sequence (MYPT1SO), or lacked only the LZ domain (MYPT1TR2). Using coimmunoprecipitation, we found that only the fragments containing the aa 888-928 sequence (MYPT1FL and MYPT1TR2) were able to form a complex with PKGIalpha in avian smooth muscle tissue lysates. Furthermore, mutations of the RK motif at aa 916-917 (R(916)K(917)) to AA decreased binding of MYPT1 to PKGIalpha in chicken gizzard lysates; these mutations had no effect on binding in chicken aorta lysates. However, mutation of R(916)K(917) to E(916)E(917) eliminated binding, suggesting that one factor important for the PKGIalpha-MYPT1 interaction is the charge at aa 916-917. These results suggest that, during cGMP-mediated signaling, aa 888-928 of MYPT1 mediate the PKGIalpha-MYPT1 interaction.
Amino Acid Motifs↗
Captopril prevents myosin light chain phosphatase isoform switching to preserve normal cGMP-mediated vasodilatation.
Congestive heart failure (CHF) is characterized by abnormal vasoconstriction and an impairment in nitric oxide (NO)-mediated vasodilatation. We have previously demonstrated that the decrease in sensitivity to NO lies at least partially at the level of the smooth muscle and is due to a reduction in the relative expression of the leucine zipper positive (LZ(+)) isoform of the myosin targeting subunit (MYPT1) of myosin light chain phosphatase. We hypothesized that since the attenuated vasodilatory response to NO in CHF has been shown to be secondary to an increased activity of the renin-angiotensin system, angiotensin converting enzyme (ACE) inhibition could affect MYPT1 isoform expression. To test this hypothesis, a rat myocardial infarction (MI) model of CHF was used; following left coronary artery ligation, rats were divided into control and captopril-treated groups. A third group of rats was given prazosin for 4 weeks. In the untreated control group, left ventricular function (LVF) was reduced at 2 weeks post-MI and remained at this level. Captopril treatment attenuated the fall in LVF. In the control aorta and iliac artery, the expression of the LZ(+) MYPT1 isoform fell 44-52% between 2 and 4 weeks post-MI, whereas in animals treated with captopril, MYPT1 isoform expression did not change. A decrease in the sensitivity to cGMP-mediated smooth muscle relaxation occurred coincident with the decrease in LZ(+) MYPT1 expression. The change in LZ(+) MYPT1 expression was not due to the decrease in afterload, as prazosin therapy produced an improvement in LVF but did not increase the relative expression of LZ(+) MYPT1 isoform. These data suggest that ACE inhibition, unique from pure afterload reduction, prevents MYPT1 isoform switching, which would preserve normal flow, or NO-mediated vasodilatation.
Animals↗
Nonmuscle myosin, force maintenance, and the tonic contractile phenotype in smooth muscle.
Recent studies have demonstrated that nonmuscle (NM) myosin II forms filaments and can generate and maintain force in smooth muscle tissue [Lofgren et al. (2003) J Gen Physiol 121:301-310; Morano et al. (2000) Nat Cell Biol 2:371-375]. To further investigate the mechanical contribution of NM myosin to force maintenance during smooth muscle contraction, we utilized a selective inhibitor of the NM myosin ATPase, blebbistatin [Straight et al. (2003) Science 299:1743-1747]. Force and myosin light chain (MLC(20)) phosphorylation were measured during KCl stimulation of small strips of intact mouse bladder and aorta at 22 degrees C. The bladder strips contracted with a typical phasic force response, characterized by a large, rapid, transient increase in force followed by a decline to a lower, steady-state level. The addition of blebbistatin did not alter the peak force, but decreased force maintenance. KCl depolarization of aortic strips resulted in a tonic contraction; force increased to a sustained steady state. Similar to the bladder tissue, blebbistatin substantially decreased the steady-state force in the aorta. Blebbistatin did not influence the MLC(20) phosphorylation transient in either tissue type. Additionally, blebbistatin did not change the maximum shortening velocity (V (max)) during KCl depolarization of the aorta. Our results also suggest that NMIIA and NMIIB isoforms are differentially expressed. The expression of NMIIA is more prominent in the bladder, while NMIIB expression is predominant in the aorta. These results suggest that NM myosin contributes to the mechanism of force maintenance in smooth muscle, and could suggest that the expression of NMIIB is a factor for determining the tonic contractile phenotype.
Animals↗
PHI-1 induced enhancement of myosin phosphorylation in chicken smooth muscle.
Herein, we provide evidence that in chicken smooth muscle, G-protein stimulation by a Rho-kinase pathway leads to an increase in myosin light chain phosphorylation. Additionally, G-protein stimulation did not increase MYPT1 phosphorylation at Thr695 or Thr850, and CPI-17, was not expressed in chicken smooth muscle. However, PHI-1 was present in chicken smooth muscle tissues. Both agonist and GTP(gamma)S stimulation result in an increase in PHI-1 phosphorylation, which is inhibited by inhibitors to both Rho-kinase (Y-27632) and (PKC) GF109203x. These data suggest that PHI-1 may act as a CPI-17 analog in chicken smooth muscle and inhibit myosin phosphatase activity during G-protein stimulation to produce Ca2+ sensitization.
Amides↗
Vascular reactivity in heart failure: role of myosin light chain phosphatase.
Congestive heart failure (CHF) is a clinical syndrome, which is the result of systolic or diastolic ventricular dysfunction. During CHF, vascular tone is regulated by the interplay of neurohormonal mechanisms and endothelial-dependent factors and is characterized by both central and peripheral vasoconstriction as well as a resistance to nitric oxide (NO)-mediated vasodilatation. At the molecular level, vascular tone depends on the level of regulatory myosin light chain phosphorylation, which is determined by the relative activities of myosin light chain kinase and myosin light chain phosphatase (MLCP). The MLCP is a trimeric enzyme with a catalytic, a 20-kDa and a myosin targeting (MYPT1) subunit. Alternative splicing of a 3' exon produces leucine zipper positive and negative (LZ+/-) MYPT1 isoforms. Expression of a LZ+ MYPT1 has been suggested to be required for NO-mediated smooth muscle relaxation. Thus, we hypothesized that the resistance to NO-mediated vasodilatation in CHF could be attributable to a change in the relative expression of LZ+/- MYPT1 isoforms. To test this hypothesis, left coronary artery ligation was used to induce CHF in rats, and both the dose response relationship of relaxation to 8-Br-cGMP in skinned smooth muscle and the relative expression of LZ+/- MYPT1 isoforms were determined. In control animals, the expression of the LZ+ MYPT1 isoform predominated in both the aorta and iliac artery. In CHF rats, LVEF was reduced to 30+/-5% and there was a significant decrease in both the sensitivity to 8-Br-cGMP and expression of the LZ+ MYPT1 isoform. These results indicate that CHF is associated with a decrease in the relative expression of the LZ+ MYPT1 isoform and the sensitivity to 8-Br-cGMP-mediated smooth muscle relaxation. The data suggest that the resistance to NO-mediated relaxation observed during CHF lies at least in part at the level of the smooth muscle and is a consequence of the decrease in the expression of the LZ+ MYPT1 isoform.
Alternative Splicing↗
Myosin heavy chain isoform expression regulates shortening velocity in smooth muscle: studies using an SMB KO mouse line.
The kinetics of smooth muscle are thought to be partially determined by the level of the expression of the 7 amino acid insert, SMB, in the myosin heavy chain, as SMB is generally expressed at higher levels in faster smooth muscle. In this study, we determined the role of this insert on shortening velocity and force regeneration following rapid reduction in muscle length (k(step)) in bladder tissue from a transgenic mouse line expressing the insert at three different levels: wild type (WT, +/+, SMB/SMB), an SMA homozygous type (SMB KO, -/-), and a heterozygous type (+/-, SMB/SMA). Smooth muscle from +/+ bladder shorten faster than both the +/- and -/- bladder smooth muscle when activated with Ca2+, consistent with SMB determining the shortening velocity of smooth muscle. The addition of Pi to the fully activated skinned bladder strips did not affect the rate of shortening for either the +/+ or -/- bladder types but did significantly decrease the rate of shortening for the +/- type. In contrast, the addition of ADP to fully Ca2+ activated bladder strips increased the rate of shortening for all three bladder types. However after thiophosphorylation, ADP slowed the shortening velocity. These data are consistent with shortening velocity being determined by the level of activation (or crossbridge attachment) in smooth muscle. The rates of force regeneration according to the k(step) protocol showed no differences between bladder types and also proved insensitive to either Pi or ADP. These data suggest that the rates of force regeneration were determined not only by the kinetics of the crossbridge cycle, but also by factors outside the contractile apparatus.
Adenosine Diphosphate↗
Unzipping the role of myosin light chain phosphatase in smooth muscle cell relaxation.
Recently, it has been hypothesized that myosin light chain (MLC) phosphatase is activated by cGMP-dependent protein kinase (PKG) via a leucine zipper-leucine zipper (LZ-LZ) interaction through the C-terminal LZ in the myosin-binding subunit (MBS) of MLC phosphatase and the N-terminal LZ of PKG (Surks, H. K., Mochizuki, N., Kasai, Y., Georgescu, S. P., Tang, K. M., Ito, M., Lincoln, T. M., and Mendelsohn, M. E. (1999) Science 286, 1583-1587). Alternative splicing of a 3'-exon produces a LZ+ or LZ- MBS, and the sensitivity to cGMP-mediated smooth muscle relaxation correlates with the relative expression of LZ+/LZ- MBS isoforms (Khatri, J. J., Joyce, K. M., Brozovich, F. V., and Fisher, S. A. (2001) J. Biol. Chem. 276, 37250 -37257). In the present study, we determined the effect of LZ+/LZ- MBS isoforms on cGMP-induced MLC20 dephosphorylation. Four avian smooth muscle MBS-recombinant adenoviruses were prepared and transfected into cultured embryonic chicken gizzard smooth muscle cells. The expressed exogenous MBS isoforms were shown to replace the endogenous isoform in the MLC phosphatase holoenzyme. The interaction of type I PKG (PKGI) with the MBS did not depend on the presence of cGMP or the MBS LZ. However, direct activation of PKGI by 8-bromo-cGMP produced a dose-dependent decrease in MLC20 phosphorylation (p<0.05) only in smooth muscle cells expressing a LZ+ MBS. These results suggest that the activation of MLC phosphatase by PKGI requires a LZ+ MBS, but the binding of PKGI to the MBS is not mediated by a LZ-LZ interaction. Thus, the relative expression of LZ+/LZ- MBS isoforms could explain differences in tissue sensitivity to NO-mediated vasodilatation.
Alternative Splicing↗
Rho signaling: agonist stimulation and depolarization come together.
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Animals↗
Creatine phosphate consumption and the actomyosin crossbridge cycle in cardiac muscles.
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.
Actomyosin↗
Force maintenance in smooth muscle: analysis using sinusoidal perturbations.
The "latch state" or force maintenance may be due to the emergence of a distinct set of dephosphorylated, slowly cycling "latch" cross-bridges, slowing of the overall cross-bridge cycling rate, or a non-cross-bridge contribution. This was investigated by sinusoidally oscillating strips of intact rabbit portal vein or aorta. Tissue strips were activated with KCl depolarization, resulting in a sustained increase of MLC(20) phosphorylation or 10 microM phenylephrine, resulting in a transient increase in MLC(20) phosphorylation. Stiffness was calculated from the force response to a small, sine-wave oscillation in muscle length (1-100 Hz). The results produced a 3-dimensional plot of stiffness versus the frequency of oscillation (Hz) versus time (s), or stiffness distribution profile. During KCl depolarization, the stiffness distribution profile displayed a shift toward lower frequencies, suggesting a general slowing in the overall cross-bridge cycling rate during force maintenance. On the other hand, phenylephrine stimulation did not display a significant change in the stiffness distribution profile, suggesting that the overall cross-bridge cycling rate did not significantly change during force maintenance.
Animals↗
The smooth muscle myosin seven amino acid heavy chain insert's kinetic role in the crossbridge cycle for mouse bladder.
The seven amino acid insert in the smooth muscle myosin heavy chain is thought to regulate the kinetics of contraction, contributing to the differences between fast and slow smooth muscle. The effects of this insert on force and stiffness were determined in bladder tissue of a transgenic mouse line expressing the insert SMB at one of three levels: an SMB wild type (+/+), an SMA homozygous type (-/-) and a heterozygous type (+/-). For skinned muscle, an increase in MgADP or inorganic phosphate (Pi) should shift the distribution of crossbridges in the actomyosin ATPase (AMATPase) to increase the relative population of the crossbridge state prior to ADP release and Pi release, respectively. Exogenous ADP increased force and stiffness in a manner consistent with increasing the Ca2+ concentration in both the +/+ and +/- mouse types. However, the -/- type showed a significantly greater increase in force than in stiffness suggesting that immediately prior to ADP release, the AMATPase either has an additional force producing isomerization state or a slower ADP dissociation rate for the -/- type compared to the +/+ or +/- types. Exogenous Pi led to a significantly greater decrease in stiffness than in force for all three mouse types suggesting that there is a force producing state prior to Pi release. In addition, the increase in Pi showed similar changes in the +/+ and -/- types whereas in the +/- type the decreases in both force and stiffness were greater than the other two mouse types indicating that the insert can affect the cooperativity between myosin heads. In conclusion, the seven amino acid insert modulates the kinetics and/or states of the AMATPase, which could lead to differences in the kinetics of contraction between fast and slow smooth muscle.
Adenosine Diphosphate↗
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.
Animals↗
The kinetic properties of smooth muscle: how a little extra weight makes myosin faster.
The contractile properties of smooth muscle (SM) are often described as fast and slow, but the molecular basis for the diversity in contractile properties has yet to be fully elucidated. Studies have shown that the differences in the contractile parameters are seen at the level of the contractile proteins. Experiments have implicated both the splicing of the SM myosin heavy chain (MHC) and the SM myosin essential myosin light chain as possible molecular determinants of the contractile properties of SM. This communication will focus on the role of the 7 aa insert in the smooth muscle MHC in determining the contractile properties of SM and the possible mechanism by which this insert could alter the kinetics of the SM actomyosin ATPase.
Actomyosin↗
Troponin T isoforms modulate calcium dependence of the kinetics of the cross-bridge cycle: studies using a transgenic mouse line.
Alternative splicing of troponin T (TnT) in striated muscle during development results in expression of different isoforms, with the splicing of a 5(') exon of TnT resulting in the expression of low-molecular-weight basic adult TnT isoforms and high-molecular-weight acidic embryonic TnT isoforms. Although other differences exist, the main differences between cardiac TnT (cTnT) and fast skeletal muscle TnT (fTnT) are in the NH(2) terminus, with fTnT being less acidic than cTnT. A transgenic mouse line expressing chicken fTnT in the heart was used to investigate the functional significance of TnT NH(2)-terminal charge differences on cardiac muscle contractility. The rates of force redevelopment (k(tr)) at four levels of Ca(2+) activation were recorded for skinned left ventricular trabeculae from control and transgenic mice. The k(tr) vs Ca(2+) relationship was different in control mice and transgenic mice, suggesting that the structure of TnT, and possibly the NH(2)-terminal region, is involved in determining the kinetics of cross-bridge cycle. These results suggest that isoform shifts in TnT may be an important molecular mechanism for determining the Ca(2+) dependence of cardiac muscle contractility.
Animals↗
Myosin light chain phosphatase: it gets around.
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Animals↗