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(Circulation Research. 2004;95:612.)
© 2004 American Heart Association, Inc.

Cellular Biology

Vascular Reactivity in Heart Failure

Role of Myosin Light Chain Phosphatase

Syed M. Karim, Albert Y. Rhee, Allison M. Given, Michael D. Faulx, Brian D. Hoit, Frank V. Brozovich

From the Departments of Physiology and Biophysics (A.Y.R., A.M.G., F.V.B.) and Medicine (Cardiology) (S.M.K., M.D.F., B.D.H., F.V.B.), Case Western Reserve University, Cleveland, Ohio.

Correspondence to Frank V. Brozovich, Department of Physiology, Case Western Reserve Univerisity, 10900 Euclid Ave, Cleveland, OH 44106-4970. E-mail Frank.Brozovich{at}case.edu

	Abstract

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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.

Key Words: cGMP • vascular function • nitric oxide • congestive heart failure

	Introduction

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Congestive heart failure (CHF) is a clinical syndrome, which is the result of systolic or diastolic ventricular dysfunction. It is associated with vascular abnormalities including a resting vasoconstriction^1,2 and resistance to nitric oxide (NO)–mediated vasodilatation.^1–3 Previous studies have demonstrated impairment in NO-mediated vasodilatation in renal and cerebral arteries in rat models of heart failure,^4,5 and similar attenuated responses to nitrates have been demonstrated in the peripheral vasculature in humans with heart failure.¹ The decrease in the sensitivity to NO has been attributed to both endothelial dysfunction^6–8 and abnormalities at the level of smooth muscle.² Nonetheless because nitrates decrease both preload and afterload, nitrates are beneficial in both the acute treatment of decompensated CHF as well as the chronic treatment of patients with CHF.^9,10 Thus understanding the mechanism for both the regulation of vascular tone and the resistance to NO-mediated vasodilatation is critical for the management of congestive heart failure.

The level of regulatory myosin light chain 20 (MLC₂₀) phosphorylation has been shown to determine the force of smooth muscle contraction¹¹ and thus vascular tone. Therefore, vascular tone is critically dependent on the activities of myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP), and steady state MLC₂₀ phosphorylation can be computed¹² as the activities of (MLCK/(MLCK+MLCP)). MLCK is a Ca²⁺-calmodulin dependent enzyme,^13,14 whereas the activity of MLCP can be both inhibited to produce Ca²⁺ sensitization (see review¹⁵), or stimulated to produce Ca²⁺ desensitization.^15,16

MLCP is a heterotrimeric enzyme (see review¹⁷) consisting of a 37-kDa catalytic subunit, a 20-kDa subunit of unknown function, and a 130/133-kDa myosin targeting subunit (MYPT1). Alternative splicing of a central and 3' exon generates four isoforms of MYPT1.^17–19 Specifically, exclusion of a 31-bp 3' exon results in a shift of the reading frame of the MYPT1 transcript to encode a C-terminal LZ motif.¹⁹ Additionally, both the activity²⁰ and regulation²¹ of MLCP has been suggested to be isoform specific.

NO-mediated vasodilatation is one of the fundamental vascular responses.^16,22 NO is known to activate the soluble pool of guanylate cyclase and result in an increase in cGMP, which in turn activates type I protein kinase G (PKGI). The activation of PKGI results in smooth muscle cell relaxation and a vasodilatation attributable to hyperpolarization,²³ a decrease in Ca²⁺ flux,^24,25 and an activation of MLCP.²⁶ The importance of cGMP-mediated activation of MLCP in smooth muscle relaxation is underscored by recent results that have demonstrated that the sensitivity of smooth muscle relaxation to cGMP is regulated by the level of expression, relative to total MYPT1 expression, of the LZ⁺ MYPT1 isoform¹⁹ and PKGI activation of MLCP requires a LZ⁺ MYPT1.²⁷

We have previously shown for MYPT1 that alternative mRNA splicing is both developmentally regulated and tissue specific, and thus so is MYPT1 isoform expression.^18,19 This could suggest that MYPT1 splicing is modulated during disease states. In this study, we determined if the resistance to cGMP-mediated vasodilatation during CHF is attributable to a change in the relative expression of LZ^+/– MYPT1 isoforms, an abnormality at the level of the smooth muscle.

	Materials and Methods

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Animal Model of Congestive Heart Failure
We used a well-accepted and studied model of CHF, the rat infarct model, which many others have demonstrated has signs of CHF including a decrease in left ventricular function (LVF), dilation of the LV, hypervolemia, pleural effusions, and ascites.^28–31 Briefly, using a surgical protocol approved by the IACUC of Case Western Reserve University, Sprague Dawley rats (Harlan, Indianapolis, Ind) were placed under general anesthesia using a combination of IP ketamine, xylazine, and acepromazine, and then intubated and placed on a mechanical ventilator (Harvard Apparatus). A left thoracotomy was performed, and the heart was exposed. The anterior coronary arteries were identified at the base of the ventricle and ligated using 6–0 suture. The chest was then closed and the animals were allowed to recover in a temperature-regulated environment under close observation before being returned to the Animal Research Center. Additionally, a group of animals underwent sham surgery, in which the procedure was identical except that only the heart was exposed before the chest was closed.

In control and postmyocardial infarction rats, left ventricular function was assessed by echocardiography at 2-week intervals. Images were collected using a cardiac ultrasound system (Acuson Sequoia) equipped with a 13-MHz linear array transducer. Two-dimension digital loop images were obtained in the parasternal long axis, parasternal short axis, and subcostal views, and measurements of diastolic wall thickness, chamber dimensions in both systole (ESD), and diastole (EDD) were made with digital calipers, as previously described.³² Fractional shortening was calculated as [((EDD–ESD)/EDD)x100], and left ventricular ejection fraction (LVEF) was estimated from wall motion and thickening.³² All other studies were performed at 8 weeks after the infarction.

Mechanical Studies
Small smooth muscle strips isolated from the aorta, iliac artery, and portal vein were used from control and CHF rats to determine the dose-response relationship to 8-Br-cGMP. The methods for tissue preparation and composition of all solutions have been described in detail.^33,34 Briefly, the smooth muscle was placed in Ca²⁺ free physiological saline solution (NaCl 140 mmol/L, KCl 4.7 mmol/L, Na₂HPO₄ 2.25 mmol/L, KCl 1.2 mmol/L, glucose 5.6 mmol/L, MOPS 2 mmol/L, and EDTA 0.5 mmol/L at a pH of 7.4), and then cleaned of connective tissue. Smooth muscle strips of approximately 600 to 800 µm in length, 200 to 600 µm in width, and 50 µm thick (muscle cross-sectional area was calculated as widthxthickness) were isolated and clamped at both ends in aluminum foil T-clips. The tissue was then skinned at room temperature for 10 minutes at pCa 9 (–log₁₀[Ca²⁺]) with 1% triton-X100 (MgATP 5 mmol/L, K₂EGTA 5 mmol/L, KMS 25 mmol/L, MgCl₂ 6.9 mmol/L, creatine phosphate 25 mmol/L, BES 5 mmol/L, and glutathione 2 mmol/L). After skinning, the smooth muscle strips were transferred to a mechanics workstation and mounted between a piezoresistive force transducer (Sensor One, San Francisco, Calif) and a length driver (Polytec PI, Auburn, Mass). The strips were stretched to L_o, the length for optimum force production, in pCa 9 relaxing solution (MgATP 5.47 mmol/L, K₂EGTA 10 mmol/L, KMS 56.5 mmol/L, MgCl₂ 7.22 mmol/L, creatine phosphate 25 mmol/L, BES 25 mmol/L, and CaCl₂ 0.02 mmol/L). The smooth muscle strips were then maximally Ca²⁺ (pCa 4) activated (MgATP 5.64 mmol/L, K₂EGTA 10 mmol/L, KMS 35.8 mmol/L, MgCl₂ 6.97 mmol/L, CP 25 mmol/L, BES 25 mmol/L, and CaCl₂ 10.3 mmol/L). After force reached a steady state, the dose-response relationship to 8-Br-cGMP (Calbiochem) added to the pCa 4 activating solution was determined.

RT-PCR
The relative level of mRNA of LZ⁺/LZ^– MYPT1 isoforms was determined using RT-PCR, as described previously.^19,35,36 The exclusion of a 31-bp 3' exon results in a shift of the reading frame of the MYPT1 transcript to encode a C-terminal LZ motif.¹⁹ In the PCR used in our experiments,¹⁹ transcripts excluding the 31-bp exon result in the amplification of a 285 bp product, whereas the inclusion of the 3' exon generates a 316-bp product. The amplified PCR products were separated using a 1.5% agarose gel and quantification of the relative expression of LZ⁺/LZ^– bands were performed using MultiAnalyst/Macintosh software.

Western Blots
Western blots were performed as previously described.^21,27 Briefly, total protein was extracted from the smooth muscle tissue samples in SDS sample buffer and resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with an acrylamide/bisacrylamide ratio of 29:1. MYPT1 was resolved using 6% gels, whereas PKGI was resolved using 10% gels, and protein loading, unless otherwise noted, was normalized for the actin band. After SDS-PAGE separation, protein bands were electrophoretically transferred to a nitrocellulose membrane (Schleicher and Schuell) in buffer containing 25 mmol/L Tris-HCl, 192 mmol/L glycine, and 10% methanol (v/v). Antibodies used were a polyclonal anti-MYPT1 (Upstate Biotechnology), a monoclonal anti-MYPT1 that detects only LZ⁺ MYPT1 isoforms (Figure 1), anti-PKGI/ß (Stressgen Biotechnologies Corp), and anti-PKGIß (Stressgen Biotechnologies Corp). Secondary antibodies specific to primary antibody were alkaline-phosphatase-conjugated. Densitometry was used to determine the intensities of the bands using an automated unit consisting of a flat bed scanner and analysis software (Amersham).

View larger version (24K): [in this window] [in a new window]   Figure 1. Specificity of the LZ+ MYPT1 Antibody. Alternative splicing of a central exon produces MYPT1 isoforms that either contain or lack a central insert (CI+/–), whereas alternative splicing of a 3' exon generates either LZ+ or LZ– MYPT1 isoforms (see text for details). Thus, alternative splicing generates four MYPT1 isoforms; CI+/LZ+, CI+/LZ–, CI–/LZ–, and CI–/LZ+. A MYPT1 protein fragment (aa 512-1030) containing both the CI and LZ was used to generate a monoclonal antibody. Four MYPT1 fragments (beginning at aa 512) were cloned, expressed, and purified. The inclusion/exclusion of the two alternatively spliced exons generated four MYPT1 protein fragments (CI+/LZ+, CI+/LZ–, CI–/LZ–, and CI–/LZ+). Western blots of the four MYPT1 fragments demonstrate that the polyclonal anti-MYPT1 antibody (Upstate Biotechnology) recognizes all four fragments (A), whereas the monoclonal anti-MYPT1 antibody did not recognize either LZ– MYPT1 fragment, but detected both LZ+ MYPT1 fragments (Panel B).

Statistics
All values reported in the text are mean±SEM and a total of n=6 to 10 animals are included in each experimental group. Differences between means were determined using an ANOVA and P<0.05 was taken as significant. There was no difference between the data for control and sham surgical animals, and the data are labeled as control.

	Results

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Left Ventricular Function
Two-dimensional echocardiography was performed on both control and CHF rats. The control animals had LVEF of 65±5%. After ligation of the LAD, the anterior wall and septum became hypokinetic to akinetic, the left ventricular end diastolic dimension increased, and the LVEF fell to 30±5% (n=20, Figure 2, Table 1). Further, the CHF animals would stand in their cage with their noses elevated, which is suggestive of orthopnea, and at the time of euthanasia had pleural effusions. These findings are consistent with the development of the clinical syndrome of CHF in the rats 8 weeks after an anterior myocardial infarction (MI).

View larger version (86K): [in this window] [in a new window]   Figure 2. LV Function in Control and CHF Rats. Representative M-mode echocardiographic image of the parasternal long axis of control and CHF rats. In the CHF rats, the LV is dilated and there is hypo- to akinesis of the septum, and additionally there was an increase in the resting heart rate.

View this table: [in this window] [in a new window]   Table 1. Left Ventricular Function

Mechanical Studies
After Ca²⁺ activation of permeabilized smooth muscle strips, force rose to a steady state in both control and CHF animals. There was no significant difference in the maximum force per cross-section between the control and the CHF rats (Table 2), suggesting that tissues were permeabilized to similar extents. In both controls and CHF animals, the aorta generated the highest force, whereas the iliac artery developed the lowest force.

View this table: [in this window] [in a new window]   Table 2. Maximal Ca2+ Activated Force

Overall, the smooth muscles from the control animals, compared with CHF animals, were significantly more sensitive to 8-Br-cGMP (Figure 3). In skinned control aortic strips, 1 nmol/L 8-Br-cGMP produced a 58±14% (n=6) relaxation compared with only 8±3% (n=5, P<0.05) in the CHF animals; in control aorta complete relaxation was produced by 10 µmol/L 8-Br-cGMP, whereas in CHF aortic strips 100 µmol/L 8-Br-cGMP only produced a 47±19% relaxation. Similarly for the iliac artery, 10 nmol/L 8-Br-cGMP completely relaxed control strips (n=6), compared with a 30±6% (n=5) relaxation in the CHF rats. Further, 10 mmol/L 8-Br-cGMP only produced a 66±12% relaxation iliac artery strips from CHF rats (Figure 3). The portal vein was relatively insensitive to 8-Br-cGMP in both control and CHF rats; 1 nmol/L 8-Br-cGMP produced a 3±1% relaxation for both the control (n=5) and CHF (n=5) animals, whereas 100 µmol/L 8-Br-cGMP produced a 52±9% relaxation in the portal vein from control rats compared with a 37±12% relaxation in CHF rats (Figure 3). Although the relaxation response of the portal vein to higher concentrations of 8-Br-cGMP was decreased in the CHF rats, these differences were not statistically significant.

View larger version (20K): [in this window] [in a new window]   Figure 3. Dose-response relationship to 8-Br-cGMP. Skinned smooth muscle strips were first maximally activated with Ca2+ (pCa 4) and after force reached a steady state, 8-Br-cGMP was added to the pCa 4 solution and the dose-response relationship of relaxation was determined. Aorta (A), portal vein (B), and iliac artery (C). Data points are mean±SEM for n=5 to 6 in control () and CHF (), respectively.

Protein Expression and Message
To determine whether CHF was associated in a change in protein expression of MYPT1 isoforms, we performed Western blots using both a polyclonal MYPT1 antibody and a monoclonal antibody selective for only LZ⁺ MYPT1 isoforms. As can be seen (Figure 4A), in smooth muscle from the CHF rats compared with control tissues for the same level of total MYPT1, the expression of the LZ⁺ MYPT1 isoform decreased markedly in the iliac artery and modestly in the aorta. The ratio of the density of LZ⁺ band/total MYPT1 band was calculated to quantify the relative expression of the LZ⁺ MYPT1 isoform. However, this quantification uses two antibodies with different affinities, and thus this ratio is only a relative measure of protein expression. In control tissues, the relative expression of the LZ⁺ MYPT1 isoform was highest in the iliac artery (Figure 4B). Compared with the control tissues, in the CHF animals, the expression of the LZ⁺ MYPT1 isoform significantly decreased (P<0.05) in both the aorta and the iliac artery, but the decrease was more pronounced (P<0.05) in the iliac artery compared with the aorta (30% versus 20%, respectively). The expression of the LZ⁺ MYPT1 isoform in the portal vein was low, and did not change after the development of CHF.

View larger version (39K): [in this window] [in a new window]   Figure 4. LZ+ MYPT1 isoform expression. A, Western blots of the aorta, portal vein, and iliac artery smooth muscle from control (left three lanes) and CHF (right three lanes) rats using antibodies against MYPT1 (top blot) and a monoclonal antibody specific for LZ+ MYPT1 (bottom blot), respectively. The arrows indicate the protein recognized by its specific antibody. In CHF, the expression of the LZ+ MYPT1 isoform decreased in both the aorta and iliac artery. B, Bar graph of the relative expression of the LZ+ MYPT1 isoform (see text for details) in control and CHF (n=3 to 6).

To confirm that this decrease in protein expression in the aorta and iliac artery was accompanied by a change in the relative mRNA levels, we used RT-PCR to determine the relative mRNA levels for LZ⁺ and LZ^– MYPT1 transcripts (Figure 5). In control animals, the expression the LZ⁺ MYPT1 transcript predominated in aorta (94±6%) and iliac artery (67±4%). Eight weeks after ligation of the anterior coronary artery in the CHF animals there was a decrease (P<0.05) in the expression of the LZ⁺ MYPT1 transcript to 79±6% in aorta and 37±5% in the iliac artery (Figure 5). Thus for both the aorta and iliac artery, CHF was associated with a significant decrease in the expression of both MYPT1 LZ⁺ transcript and protein.

View larger version (24K): [in this window] [in a new window]   Figure 5. Relative mRNA Levels. Bar graph of the RT-PCR results showing relative mRNA level of the LZ+ MYPT1 transcript (n=5 to 6) in aortic and iliac artery smooth muscle of control and CHF rats. Inset, Representative agarose gel showing transcript pattern of MYPT1 in aortic (A) and iliac artery (IA) smooth muscle in control and CHF rats. Lower band is 3' exon out, or the LZ+ MYPT1 transcript.

We also used Western blots to determine whether a change in the expression of PKGI or a switch in PKGI isoform expression, from PKGI to PKGIß, accompanied CHF. Western blots using a nonspecific PKGI/ß antibody showed the presence of only a single protein band at 78 kDa in smooth muscle from both control and CHF rats (Figure 6). The intensity of this band was similar (P>0.05) in control and CHF tissues, suggesting that CHF is not accompanied by a change in total PKGI expression. In addition in both control and CHF smooth muscle, a PKGIß-specific antibody did not detect protein, which suggests that PKGI expression is exclusively PKGI.

View larger version (36K): [in this window] [in a new window]   Figure 6. PKGI expression. Western blots of the aorta, portal vein, and iliac artery smooth muscle from control (right three lanes) and CHF (left three lanes) rats using a nonspecific PKGI/ß antibody (top) and PKGIß specific antibody (bottom). A positive control for PKGIß is in the lane labeled NM cells. Arrows indicate the protein recognized by its specific antibody. The nonspecific PKGI antibody detected a single protein band and the intensity of this band is similar (P>0.05) in smooth muscle from control and CHF rats (n=5 to 6). There was no protein detected with the specific antibody to PKGIß, indicating that expression is exclusively PKGI in smooth muscle isolated from both control and CHF rats.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
NO-mediated vasodilatation is a fundamental response of the vasculature.^16,22 The basic mechanism governing NO-mediated smooth muscle relaxation has been delineated; NO is known to stimulate guanylate cyclase to increase cGMP which in turn activates PKGI.³⁷ PKGI has multiple targets in smooth muscle, which all result in smooth muscle relaxation by different mechanisms including phosphorylation of the maxi K⁺ channel to produce hyperpolarization,²³ both Ca²⁺ channels²⁵ and the SR²⁴ to decrease intracellular Ca²⁺, and an activation of MLCP.²⁶

However, there is diversity in the response of smooth muscle to NO in normal tissues,^38–40 as well as a blunted response in CHF.^1–8 The mechanism responsible for differential sensitivity to NO has been suggested to be attributable to changes in the relative expression of LZ⁺/LZ^– MYPT1 isoforms.²⁷ Surks et al^26,41 have demonstrated that PKGI activates MLCP phosphatase activity, and postulated that this was attributable to a LZ-LZ interaction of the NH₂-terminal LZ of PKGI with the COOH-terminal LZ of MYPT1. Moreover, we have previously demonstrated that the sensitivity of skinned smooth muscle to cGMP-mediated relaxation correlates with the expression of the LZ⁺ MYPT1 isoform.¹⁹ Thus, alternative MYPT1 splicing to produce changes in the relative expression of LZ⁺/LZ^– MYPT1 isoforms could explain a decrease in sensitivity to NO-mediated vasodilatation in CHF.

In our animals 8 weeks after anterior coronary artery ligation, echocardiography (Figure 2) demonstrated a significant impairment of LV systolic function and dilatation of the left ventricle (Table 1). Ligation of the LAD to produce an anterior myocardial infarction is well accepted to produce a model of CHF and LV remodeling in rats.^28–31,42 The alterations in LVF observed in our CHF animals is similar to that demonstrated by others.^28–31,42

In the rats used in this study, CHF was associated with a change in the relative expression of LZ⁺/LZ^– MYPT1 isoforms with a significant decrease in the expression at the protein level of the LZ⁺ MYPT1 isoform in both the aorta and the iliac artery (Figure 4). The decrease in LZ⁺ MYPT1 isoform expression was more prominent in the more distal vessel (iliac artery), compared with a conduit vessel (aorta), and could suggest that the modulation of LZ⁺ MYPT1 isoform expression in resistance vessels could be even more dramatic. Furthermore, the sensitivity to cGMP-mediated smooth muscle relaxation (Figure 3) scaled with the relative expression of the LZ⁺ MYPT1 isoform (Figure 4). The expression of the LZ⁺ MYPT1 isoform and sensitivity to cGMP-mediated relaxation listed from highest to lowest is control iliac artery, control aorta, CHF iliac artery, CHF aorta, and portal vein. Further the decrease in LZ⁺ MYPT1 isoform expression in both the aorta and iliac artery correlates with the decrease in the sensitivity to cGMP mediated smooth muscle relaxation. Thus, similar to normal tissues,¹⁹ the sensitivity to cGMP-mediated relaxation is determined, at least in part, by the expression of the LZ⁺ MYPT1 isoform. The sensitivity to cGMP-mediated smooth muscle relaxation was low in the portal vein consistent with the expression of the LZ⁺ MYPT1 isoform, but similar to that observed in the CHF aorta. These data could suggest that there could be a threshold level of LZ⁺ expression before a tissue is sensitive to cGMP.

PKGI is expressed in three different isoforms (PKGI, PKGIß, and PKGII) in mammalian smooth muscles,²⁶ and this group has suggested that only PKGI, but not PKGIß, dimerizes with the MYPT1 to activate MLCP activity. We demonstrated that compared with control tissue, CHF was neither associated with a change in the total PKGI expression nor a change in PKGI isoform expression, which is exclusively PKGI in both control and CHF smooth muscle (Figure 6). These data rule out the possibility that the observed decrease in sensitivity to cGMP-mediated smooth muscle relaxation in the present study was attributable to a change in PKGI expression. In addition, our experiments were performed in skinned smooth muscle, and thus we can also rule out neurohormonal activation, differences in resting membrane potential or differences in Ca²⁺ flux as contributors to the decrease in sensitivity to cGMP.

Using short GST fusion proteins, Surks et al⁴¹ have shown that MYPT1 is able to dimerize with PKGI, only if both MYPT1 and PKGI express a LZ. However, we have recently shown that both full-length avian LZ^– and LZ⁺ MYPT1 isoforms will bind PKGI,²⁷ suggesting that the PKGI-MYPT1 interaction is not mediated by the C-terminal LZ domain of MYPT1, but rather, possibly mediated by a MYPT1 coiled-coil domain present at aa 888–928.⁴³ Although the LZ is not required for the interaction of MYPT1 with PKGI, we did demonstrate that a LZ⁺ MYPT1 isoform is required for PKGI to activate MLCP activity.²⁷ Although the exact mechanism by which PKGI activates MLCP activity has yet to be elucidated, it could be attributable to coiled-coil interaction of PKGI with MYPT1^27,43 and subsequent MYPT1 phosphorylation.^26,27,41 Nonetheless, the decrease in LZ⁺ MYPT1 expression observed during CHF in the present study would suggest that the decrease in sensitivity to NO-mediated vasodilatation observed in this clinical syndrome lie at least partially at the level of the smooth muscle and is a consequence of the decrease in LZ⁺ MYPT1 expression.

Most effective therapies that result in a decrease in mortality in patients with CHF are aimed at the vasculature,^1,10 which suggests that there is an underlying abnormality of vascular function associated with this clinical syndrome. However until the present study, no data existed to suggest a possible molecular mechanism for the abnormality of vascular function. Others⁵ have demonstrated a decrease in the sensitivity of smooth muscle relaxation to nitroprusside in the rat infarct model of CHF, suggesting that in this model there is an abnormality in NO signaling at the level of the smooth muscle, independent of endothelial function. Additionally, the skinned smooth muscle strips used in the present study lack an endothelium and thus the decrease in sensitivity to cGMP mediated relaxation in the present study, reflects the abnormality in NO signaling in this animal model of CHF. Further, we have previously shown that cGMP/PKGI-mediated activation of MLCP activity requires the expression of a LZ⁺ MYPT1 subunit.²⁷ Thus during the clinical syndrome of CHF, the data in the present article suggest that the resistance to NO-mediated vasodilatation lie at least in part at the level of the smooth muscle and is a consequence of a modulation of MYPT1 LZ^+/– isoform expression, and specifically attributable to a decrease in the expression of LZ⁺ MYPT1 isoform.

An increase in blood flow is known to increase the shear stress on the endothelial cells and stimulate the production of NO, which stimulates PKGI and a resulting increase in cGMP, which activates PKGI and results in vasodilatation. Our results demonstrate that the vessel’s sensitivity to NO-mediated vasodilatation is determined, in part, by the relative expression of the LZ⁺ MYPT1 isoform. Thus, the decrease in LZ⁺ MYPT1 expression observed in our animal model of CHF would thus result in a decrease in the sensitivity to NO, and would be expected to blunt flow-mediated vasodilatation. This would increase vascular tone and thus could produce a resting vasoconstriction, another vascular abnormality associated with the clinical syndrome of CHF.

The signaling pathway to explain a change in LZ^+/– MYPT1 isoform expression both during development¹⁹ and CHF (present study) is unknown. However, pharmacologically targeting this pathway with small molecule inhibitors or other therapeutic agents aimed at blocking MYPT1 isoform switching or directly activating the MLCP, could potentially open a new, and possibly more effective avenue for treating patients with CHF.

	Acknowledgments

 
This study was supported by grants from the NIH (HL69894 to F.V.B.) and T32 HL07887 (to S.M.K.). We would like to acknowledge Dr Ozgur Ogut for his critical reading and comments on the text, and Dr Frank Chen for his technical assistance.

	Footnotes

 
Original received June 9, 2004; resubmission received July 15, 2004; revised resubmission received August 6, 2004; accepted August 10, 2004.

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