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(Circulation Research. 1995;77:114-120.)
© 1995 American Heart Association, Inc.

Articles

₁-Adrenergic Receptor Stimulation Decreases Maximum Shortening Velocity of Skinned Single Ventricular Myocytes From Rats

Kevin T. Strang, Richard L. Moss

From the Department of Physiology, School of Medicine, University of Wisconsin, Madison, Wis.

Correspondence to Kevin T. Strang, Department of Physiology, 1300 University Ave, Madison, WI 53706.

	Abstract

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Abstract ₁-Adrenergic agonists have negative inotropic effects on mammalian myocardium under some conditions, and biochemical experiments measuring the Ca²⁺-activated actomyosin ATPase activity of myofibrillar preparations suggest that this may result from a decrease in cross-bridge cycling rate caused by phosphorylation of myofilament proteins. Experiments with intact ventricular preparations, however, have failed to demonstrate a mechanical manifestation of a decrease in cycling rate. The present study examined the effect of ₁-adrenergic receptor stimulation on maximum shortening velocity in skinned single ventricular myocytes from rats. Enzymatically isolated myocytes were incubated with the ß-receptor antagonist propranolol in the presence or absence of the ₁-adrenergic receptor agonist phenylephrine and were then rapidly skinned to preserve the phosphorylation state of myofilament proteins. The velocity of unloaded shortening (V_o) was determined by use of the slack-test method and compared between skinned control and phenylephrine-treated cells. The relationship between isometric tension and [Ca²⁺] was also assessed for each myocyte. V_o was significantly lower in the ₁-adrenergic receptor agonist–treated cells than in the control cells, but there was no effect on Ca²⁺ sensitivity of isometric tension. In addition, the myosin heavy chain isoform composition accounted for a significant amount of the variation in V_o within the treatment groups. On the basis of these and previous results we propose that ₁-adrenergic receptor stimulation inhibits cross-bridge cycling rate at the level of myofilament proteins by a mechanism that may involve phosphorylation of troponin I by protein kinase C.

Key Words: ₁-adrenergic receptor • cardiac myocyte • shortening velocity • phosphorylation

	Introduction

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Stimulation by ₁-adrenergic receptors is generally associated with positive inotropy in mammalian ventricular myocardium,^{1 2 3} yet under some experimental conditions depression of contractile properties is observed.^{4 5 6 7} It has been suggested that when it occurs, this negative inotropy results from a decrease in cross-bridge cycling kinetics mediated by myofilament protein phosphorylation by protein kinase C (PKC).⁸ Phosphorylation of reconstituted cardiac actomyosin and myofibrillar preparations with PKC reduces the maximum Ca²⁺-stimulated Mg²⁺-ATPase activity,^{8 9} implying that the rate of transition between two or more steps in the cross-bridge cycle is reduced. Such a mechanism could account for the observation that ₁-adrenergic receptor stimulation prolongs the isometric twitch in intact rabbit papillary muscle without a corresponding increase in the duration of the Ca²⁺ transient.² A reduction in the rate of cross-bridge steps leading to detachment of myosin heads from actin could result in an increase in the duration of a twitch.

In contrast, other experiments suggest that ₁-adrenergic receptor stimulation has no influence on cross-bridge cycling kinetics. Adrenaline and propranolol treatment of rat papillary muscle during isometric Ba²⁺-stimulated contractures caused no change in the frequency of minimum stiffness, a mechanical property thought to reflect the overall rate of cross-bridge cycling.¹⁰ Similarly, the time to peak rate of rise of isometric twitch force and the time constant of twitch relaxation were unchanged by ₁-adrenergic receptor stimulation in isolated rat papillary muscles,¹¹ from which finding it was concluded that ₁-adrenergic receptor stimulation does not alter cross-bridge cycling rate at the level of myofilament proteins. The reason for the discrepancy between these and the previous results is not clear at present. Frequency of minimum stiffness may have been unaffected because most isoforms of PKC are Ca²⁺ dependent, and conducting those experiments under Ca²⁺-free conditions may have inhibited normal substrate phosphorylation. Furthermore, the relative importance of Ca²⁺ handling and cross-bridge kinetics in determining the twitch characteristics of intact myocardium is not presently understood.

To further address this issue, we compared the velocity of unloaded shortening (V_o) between chemically skinned control rat ventricular myocytes and myocytes from the same whole-heart preparation that were treated with the ₁-adrenergic receptor agonist phenylephrine and the ß-receptor blocker propranolol prior to skinning. V_o is thought to be determined by the rate of detachment of myosin heads from actin,¹² and biochemical measurements suggest that ADP dissociation from myosin is sufficiently slow to be the rate-limiting step in cross-bridge detachment.¹³ Therefore, changes in V_o should manifest changes in the kinetics of this step of the cross-bridge cycle. The relationship between isometric tension and [Ca²⁺] was determined in the same cells in order to assess the effect of ₁-adrenergic receptor stimulation on Ca²⁺ sensitivity of tension. We found that ₁-adrenergic receptor stimulation significantly reduced V_o in treated cells compared with control cells from the same preparation. In addition, the relative proportion of fast and slow myosin heavy chain (MHC) isoforms was a significant determinant of V_o. There was no difference in Ca²⁺ sensitivity of isometric tension between control and agonist-treated groups. We conclude that ₁-adrenergic receptor stimulation decreases the rate of cross-bridge detachment by a mechanism that probably involves phosphorylation of troponin I by PKC.

	Materials and Methods

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Single ventricular myocytes were obtained by enzymatic digestion from female Sprague-Dawley rats as previously described.^{14 15} Cells were divided into two aliquots. Half were resuspended in oxygenated 1 mmol/L Ca²⁺ Ringer's solution containing (mmol/L) MgCl₂ 1.2, CaCl₂ 1, KCl 4.8, NaCl 118, KH₂PO₄ 2, pyruvate 5, glucose 11, insulin 1, and HEPES 25 (pH 7.4) and the ₁-adrenergic receptor agonist phenylephrine (10 µmol/L) and the ß-receptor antagonist propranolol (1 µmol/L). The remaining cells were resuspended in 1 mmol/L Ca²⁺ Ringer's with propranolol only. Propranolol alone does not alter Ca²⁺ sensitivity of isometric tension or V_o in this preparation (K.T.S., unpublished observation, 1994, and Reference 15¹⁵ ). Both treatment groups were incubated for 5 minutes at 37°C and then rapidly permeabilized by resuspension for 6 minutes at 22°C in a relaxing solution containing (mmol/L) free Mg²⁺ 1, KCl 100, EGTA 2, ATP 4, and imidazole 10 (pH 7.0) and 0.3% Triton X-100 (Pierce). This procedure preserves myofibrillar proteins in a given phosphorylation state by quickly removing soluble and membrane-bound kinases and phosphatases.^{15 16} The skinned cells were finally washed twice in fresh relaxing solution and stored on ice until use. All relaxing and activating solutions had an ionic strength of 180 mmol/L.

Single cells were attached with silicone adhesive (Dow Corning) to glass micropipettes on the stage of a Zeiss inverted microscope modified for temperature control, as previously described¹⁷ (Fig 1). In brief, one pipette was fixed to a piezoelectric translator (Physik Instrument Co) and the other to a force transducer (model 403, which has a sensitivity of 20 mV/mg and resonant frequency of 300 Hz, Cambridge Technology), both of which were mounted on micromanipulators (Narishige). The output signal from the force transducer was amplified 10-fold and then input to an oscilloscope (model NIC-310, Nicolet Instrument Corp) for storage on magnetic disk and subsequent analysis. The piezoelectric device was driven by a bipolar operational power supply/amplifier (Kepco Inc) that was linear to ±50 µm at a calibration of 0.054 µm/V. The amplifier output signal was monitored on a second channel of the oscilloscope. The amplifier was driven by voltage command signals from a pulse interval generator (World Precision Instruments Inc). Sarcomere length (SL) and cell width were monitored and recorded on videotape by use of a video camera (model WV-B1600, Panasonic) and a VHS recorder (model HR-s6600u, JVC). We measured cell width and depth after each experiment by detaching one pipette and rotating the cell 90° with the remaining pipette. The calculation of cross-sectional area for all cells was based on the assumption of an elliptical cross section.

View larger version (118K): [in this window] [in a new window]   Figure 1. Photomicrographs show a single cardiac myocyte while relaxed (A) and during maximal activation (B). A, Cell length was 138 µm between pipette tips and sarcomere length was 2.29 µm. B, Sarcomere length was 2.23 µm. Resting tension was 3.4 kN/m2 and maximum active force was 46.1 kN/m2.

Ca²⁺ sensitivity of isometric tension was determined for control and ₁-adrenergic receptor–stimulated cells as follows. Isometric tension was measured during maximal activation (pCa 4.5) at the beginning, middle, and end of each experiment to assess the performance of the preparation; data were discarded if maximum tension (P_o) declined by >20% during the experiment. In between, tension was measured at varying submaximal pCa values and expressed relative to the maximum tension (P_rel). P_o was interpolated between the three maximal tension measurements to account for changes in the preparation with time. The data were analyzed by least-squares regression with the Hill equation, log[P_rel/(1-P_rel)]=n(log[Ca²⁺]+k), where n is the Hill coefficient and k is the intercept of the fitted line with the x axis, which corresponds to the [Ca²⁺] at half-maximal isometric tension (pCa₅₀). By use of the constants derived from the Hill equation, tension-pCa curves were fit by computer with the equation P_rel=[Ca²⁺]ⁿ/(kⁿ+[Ca²⁺]ⁿ).

SL was initially set at 2.30 µm and was monitored during activation. Cells were considered too compliant and were thus discarded if SL varied by >0.2 µm between relaxed and maximally activated conditions. The experimental chamber was cooled to 15°C by use of DC-powered thermoelectric devices (Cambion, Midland-Ross Co), which in turn were cooled by circulating water.

V_o was measured during maximal activation with the slack-test method,¹⁸ as illustrated in Fig 2. Once steady tension was achieved, cells were slackened by varying amounts from an initial SL of 2.3 µm, and the time required to take up the slack was measured from the beginning of the length step to the onset of tension redevelopment. The maximum step size imposed was such that cells were not allowed to shorten to an SL of <1.8 µm, at which point interference from restoring forces is likely to occur.^{19 20} The time point at which tension redeveloped was determined graphically by hand-fitting a line through the tension baseline and determining its intersection with a line drawn through the initial portion of the tension rise. Length change as a fraction of initial muscle length (ML) was plotted versus the duration of unloaded shortening. The slope of this plot was determined by linear regression and recorded as V_o.¹⁸ Criteria for discarding cells because of excessive compliance or loss of tension were as noted above. In addition, data from a given cell were not used if less than four data points were obtained.

View larger version (15K): [in this window] [in a new window]   Figure 2. Graphs show slack-test method of determining unloaded shortening velocity (Vo). A, Graph shows cell length versus time (top) and unexpanded force versus time (bottom); data were obtained during four differently sized slack steps (17.2%, 18.8%, 20.3%, and 21.9% of muscle length [ML]) in a control cell. B, Tracings were expanded, and straight lines were fit by eye through the baseline and the initial portion of rising tension. C, Slack-test plot shows the data for all slack steps in this cell: Vo was 0.98 ML/s; 55% of the myosin heavy chain protein was of the ß type.

To account for differences in V_o that could arise from myosin isoform differences between cells,²¹ the relative proportions of fast and slow MHCs were determined for individual cells by use of SDS-PAGE as previously described.²⁰ To account for variations between individual rat heart preparations, pCa₅₀ and V_o from same-day control and ₁-adrenergic receptor–stimulated cells were paired and analyzed by use of Student's paired t test. pCa₅₀ and V_o were both determined in some cells, and in others only the pCa₅₀ was measured. Differences were deemed significant for values of P<.05. Chemicals were purchased from Sigma Chemical Co unless otherwise noted.

	Results

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The preparation used in these experiments had low end-compliance, because SL shortened by an average of only 0.05 µm (ie, 2%) when cells were transferred from relaxing solution to maximally activating solution (Fig 1). Average dimensions and force characteristics for all cells studied (n=28) were as follows: cell length at SL of 2.3 µm, 107.6±19.7 µm; cell depth/width, 0.80; cross-sectional area, 249.7±142 µm²; maximal force, 43.9±13.9 kN/m²; and resting tension at SL of 2.3 µm, 4.24±1.6 kN/m². There were no differences in cellular dimensions between experimental groups.

₁-Adrenergic receptor stimulation had no effect on steady state isometric tension. P_o was 45.35±15.06 kN/m² in control cells and 42.63±13.17 kN/m² in agonist-treated cells. There was also no systematic difference in the isometric tension–pCa relationships (Fig 3); thus, tension at submaximal [Ca²⁺] was also unaltered. The lack of effect on the pCa₅₀ (Table) implies that Ca²⁺ sensitivity of tension is not affected by the agonist-treatment and rapid skinning protocol. The Hill coefficient (n) was also not significantly different between the two groups (2.59±0.53 in control and 2.67±0.93 in ₁-adrenergic receptor–stimulated cells), suggesting that the apparent molecular cooperativity of tension development was unaffected by ₁-adrenergic receptor agonist stimulation.

View larger version (16K): [in this window] [in a new window]   Figure 3. Graph shows Ca2+ sensitivity in representative control and phenylephrine-treated cells. Hill plot analysis yielded pCa50 values of 5.56 and 5.57 in the control cells and 1-adrenergic receptor–stimulated cells, respectively; the Hill coefficients were 2.77 and 2.78.

View this table: [in this window] [in a new window]   Table 1. Effect of 1-Adrenergic Stimulation on Contractile Properties of Rat Skinned Single Ventricular Myocytes

The slack-test protocol yielded reproducible linear plots of length change versus duration of unloaded shortening, with r>.95 for all cells (Figs 2 and 4). The value of V_o determined from the slope of these plots was 2 ML/s at 15°C for control cells containing predominantly -MHC. Assuming a Q₁₀ for V_o of 4.6,²² this value is within the ranges previously reported for rat myocardium (2.25 ML/s at 15°C,¹⁵ 2.6 ML/s at 20°C,²² 2.2 ML/s at 5.5°C,²³ 4.2 ML/s at 17°C,²⁴ and 4.46 ML/s at 22°C²⁵ ). In general, it was not possible to obtain slack records for length steps <17% of ML. At smaller step lengths, there were large noise oscillations in the force record, and steady zero-baseline tensions were not apparent. This is presumably because these small steps did not completely unload the force borne by passive elastic elements, which increases significantly at SLs of >2.0 µm.

View larger version (19K): [in this window] [in a new window]   Figure 4. Graphs show unloaded shortening velocity (Vo) in control and 1-adrenergic receptor–stimulated cells. Expanded force versus time tracings from a control cell (A) and a cell from the same preparation that was treated with phenylephrine and propranolol (B) demonstrate that 1-adrenergic receptor stimulation increases the time required to take up a given amount of slack. Slack steps (percent muscle length [ML]) and duration of unloaded shortening (time in milliseconds [ms]) for the three control tracings (A) were, for L=18.0, 31.2 ms; for L=19.8, 45.0 ms; and for L=21.7, 63.0 ms. In the 1-adrenergic receptor–stimulated cell (B), slack steps and duration were, for L=17.9, 33.6 ms; for L=20.1, 58.6 ms; and for L=21.7, 82.6 ms. C, Slack plot for the same two cells, including all slack steps. Vo determined from the slopes of the regression lines was 1.19 ML/s in the control cell and 0.73 ML/s in the 1-adrenergic receptor–stimulated cell.

Further evidence of significant passive elastic force in this preparation comes from the elevated y intercept of the slack plot, which averaged 14% of ML. It was demonstrated in skeletal muscle that the y intercept of the slack plot is in part determined by the extent of rapid initial shortening, which varies as a function of resting tension.²⁶ Consistent with this, an association between resting tension and the intercept of the slack plot was recently demonstrated in single cardiac myocytes after phosphorylation by ß-adrenergic agonists.¹⁵ Interestingly, in the present study the y intercept was significantly higher in phenylephrine-treated cells (16±1% of ML) than in control cells (12±2% of ML), suggesting that resting tension may be increased by ₁-adrenergic receptor stimulation (Fig 4C). Support for this possibility comes from experiments in which a decrease in resting SL was observed in intact ventricular myocytes stimulated with phenylephrine and propranolol.^{27 28} In the present experiments, however, we did not detect a significant increase in resting tension at SL of 2.3 µm in the ₁-adrenergic receptor–stimulated group. Although it is not clear why this was the case, it is possible that a difference may have been obscured by cell-to-cell variability or error in the determination of cross-sectional area.

The slope of the slack plot (V_o) was reduced by an average of 33% in myocytes treated with phenylephrine and propranolol prior to rapid skinning compared with propranolol-treated control cells (Fig 4 and Table). It is thus apparent that myofilament proteins in the ₁-adrenergic receptor–stimulated cells were modified such that the rate of cross-bridge cycling at maximal [Ca²⁺] was reduced. The difference in V_o was highly significant when cells were paired by preparation (P<.0001). In addition, the mean V_o values from the two groups were significantly different (P<.02), despite a significant amount of variability within each group (Table).

The MHC isoform composition is known to be a major determinant of cross-bridge cycling rate in cardiac muscle,²¹ so to further investigate the variability in V_o between cells within each of the treatment groups, we measured the relative proportions of fast () and slow (ß) MHC isoforms in each individual myocyte. Fig 5 demonstrates that the two MHC isoforms are easily distinguishable by SDS-PAGE and laser scanning densitometry and that individual cells varied considerably in MHC isoform composition. Plotting V_o as a function of the proportion of ß-MHC revealed that MHC isoform composition accounted for a significant portion of the variability in V_o within the treatment groups (Fig 6). Regression analysis indicated that V_o decreased by 0.8% for a 1% increase in the amount of ß-MHC isoform in both treatment groups and that MHC isoform content accounted for 79% of the variation in the control group and 89% of the variation in the ₁-adrenergic receptor–stimulated group.

View larger version (14K): [in this window] [in a new window]   Figure 5. Myosin heavy chain (MHC) region of silver-stained SDS-PAGE gel and corresponding laser densitometric scans from four individual myocytes. The areas under the peaks were integrated and the fractions of fast () and slow (ß) MHC were calculated. In these cells, ß-MHC was 0%, 19%, 51%, and 57% of total MHC protein.

View larger version (34K): [in this window] [in a new window]   Figure 6. Plot shows unloaded shortening velocity versus myosin heavy chain (MHC) isoform composition. Least-squares regression lines fit to the data were as follows: control, unloaded shortening velocity (Vo, in muscle lengths [ML] per second)=2.02-0.016(%ß-MHC); phenylephrine, Vo=1.37-0.010(%ß-MHC).

To verify that the reduction in V_o was due specifically to stimulation of ₁-adrenergic receptors, additional experiments were performed in which myocytes containing predominantly -MHC were treated with phenylephrine (10 µmol/L) in the presence of prazosin (10 µmol/L), an ₁-adrenergic receptor blocker. V_o was 2.30±0.23 ML/s (n=5) in these cells, not significantly different from control cells with similar MHC composition (2.06±0.32 ML/s, n=5). V_o in both the control and the prazosin-treated cells, however, was significantly faster than in cells treated with phenylephrine in the absence of prazosin (V_o=1.20±0.23 ML/s, n=7; P=.002 versus control, P<.0001 versus prazosin).

	Discussion

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The intracellular mechanisms mediating the effects of ₁-adrenergic receptor stimulation are complex, as evidenced by the fact that ₁-adrenergic receptor agonists induce positive inotropy in some circumstances and induce negative inotropy in others. This variable response may in part arise from differences in the dynamics of Ca²⁺ handling and myofilament protein phosphorylation under different experimental conditions. For example, at low stimulation frequencies (<1 Hz) under control conditions, ₁-adrenergic receptor agonists have a positive inotropic effect associated with an increase in the amplitude of the Ca²⁺ transient.^{2 7} Higher [Ca²⁺]_i increases twitch force directly by increasing both the number of cross-bridges and the rate at which they are recruited to force-generating states²⁹ but may also increase the phosphorylation state of myosin light chain 2 (MLC₂) through Ca²⁺-calmodulin–mediated activation of MLC kinase.³⁰ Phosphorylation of MLC₂ is known to increase the Ca²⁺ sensitivity of the myofilaments,³¹ the rate of tension generation,³² and the maximum Ca²⁺-activated actomyosin ATPase activity,³³ all of which could influence the tension and kinetics of a twitch. In contrast, a sustained negative inotropic effect results from ₁-adrenergic receptor stimulation at higher electrical stimulation frequencies (>1.5 Hz),⁵ when ß-adrenergic receptors are simultaneously activated,^{6 34 35} or when transient outward currents are blocked with 4-aminopyridine.⁷ In each of these three conditions, time-averaged [Ca²⁺]_i was already significantly elevated by other processes before ₁-adrenergic receptor agonist application. This most likely reduces or eliminates any effects of ₁-adrenergic receptor stimulation that are mediated by alterations in Ca²⁺ handling and could therefore unmask effects on cross-bridge cycling kinetics.

The present experiments directly examined the effects of ₁-adrenergic receptor stimulation in the absence of changes in Ca²⁺ transients. During activation with agonist, the cells were incubated in Ringer's solution containing 1.0 mmol/L Ca²⁺ but were not stimulated. These conditions provide physiological resting [Ca²⁺]_i but prevent changes in myofilament protein phosphorylation caused by changes in intracellular Ca²⁺ transients or time-averaged [Ca²⁺]_i.³⁶ Furthermore, the mechanical measurements were performed on skinned cells, permitting control of the intracellular Ca²⁺ bathing the myofilaments. The methods allow an assessment of the effects of ₁-adrenergic receptor stimulation on cross-bridge function without the direct or indirect influence of alterations in Ca²⁺ handling. These conditions may have relevance to the in vivo situation, in which cardiac ₁-adrenergic receptor stimulation occurs at contraction frequencies of >1.5 Hz (at least in small mammals³⁷ ) and ß-adrenergic receptors are simultaneously activated by norepinephrine.²

Our results indicate that ₁-adrenergic receptor stimulation decreases the rate of cross-bridge cycling in ventricular myocardium, and they are consistent with results of previous studies suggesting that PKC-mediated phosphorylation slows cross-bridge cycling. Treating intact ventricular preparations with ₁-adrenergic receptor agonists or phorbol esters results in translocation of PKC to the myofilaments³⁸ and increased phosphorylation of troponin I and C protein.³⁹ In other experiments, PKC treatment of reconstituted actomyosin preparations reduced the maximum Ca²⁺-activated ATPase activity by 32% to 55%.⁸ Similarly, maximum ATPase activity was reduced by 25% in myofibrils isolated from ventricular myocytes that were stimulated with a PKC-activating phorbol ester and were then rapidly skinned.³⁹ The magnitude of these effects on ATPase is similar to the reduction in V_o observed in the present study (33%). This agreement is not surprising given that the ADP-release step of the cross-bridge cycle is thought to be rate limiting both for actomyosin interaction in solution and for V_o.¹³ Taken together, these results indicate that one component of the response to ₁-adrenergic receptor stimulation is reduction of the rate of cross-bridge dissociation, an effect that is mediated by PKC phosphorylation of myofilament proteins.

Although both C protein and troponin I are likely to be phosphorylated by PKC under the conditions used in the present study,³⁹ it is improbable that C-protein phosphorylation is important in mediating the reduction in V_o. Both proteins are also phosphorylated by the principal second messenger of the ß-adrenergic pathway, cAMP-dependent protein kinase (PKA),³⁹ yet the mechanical effect of ß-adrenergic stimulation is the opposite: V_o is increased by 40%.¹⁵ Phosphopeptide mapping and autoradiography revealed that PKA and PKC phosphorylate the same sites on C protein to similar extents, whereas troponin I is phosphorylated at distinct sites by the two kinases.³⁹ Taken together, these results are consistent with earlier work showing that phosphorylation of C protein does not affect actomyosin ATPase activity⁴⁰ and suggest that troponin I phosphorylation at specific sites may modulate cross-bridge kinetics in mammalian cardiac muscle.

The correlation between V_o and MHC isoform content observed in the present study confirms previous reports that have demonstrated, by a variety of methods, that MHC isoform content is a primary determinant of the rate of cross-bridge cycling. Ca²⁺-activated actomyosin ATPase is 30% to 50% lower in rat ventricular muscle with predominantly ß-MHC compared with that with -MHC.^{23 41 42} Likewise, muscle and protein preparations with ß-MHC cycle 40% to 50% slower than those with -MHC as assessed by V_o,²³ the in vitro motility assay,⁴¹ and frequency of minimum stiffness.¹⁰ In the present study, V_o decreased by 30% as the ß-MHC isoform increased from 0% to just over 50% in both control and phenylephrine-treated groups (Fig 6).

Possible effects of ₁-adrenergic receptor stimulation on Ca²⁺ sensitivity of tension and the mechanisms by which any changes may occur are controversial. Some studies with intact myocytes indicate that alkalinization of the cytosol leads to an increase in sensitivity,^{28 43} while others suggest that phosphorylation of a myofilament protein, most likely MLC₂, results in increased sensitivity.⁴⁴ However, if this Ca²⁺-sensitization effect is a MLC₂ phosphorylation–mediated phenomenon, it is not clear whether alteration in Ca²⁺-calmodulin activation of MLC kinase is responsible³¹ or whether some specific PKC isoform might be involved.⁴⁵ More importantly, it is not even known whether myofilament sensitization (by whatever mechanism) is associated only with conditions in which ₁-adrenergic receptor stimulation enhances inotropy or occurs in all circumstances. In the present experiments there was no effect on pCa₅₀, and we can therefore conclude only that under our experimental conditions no myofilament proteins were phosphorylated, which might influence Ca²⁺ sensitivity of tension.

	Acknowledgments

 
This study was supported by a grant from the National Institutes of Health (HL-25861) to Dr Moss and an American Heart Association (Wisconsin Affiliate) Predoctoral Fellowship to Dr Strang. The authors thank Scott Stoker and Shermini Saini for their assistance with the experimental preparation and Dr James Graham for performing the SDS-PAGE on single myocytes.

Received June 7, 1994; accepted March 31, 1995.

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