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Am J Physiol Heart Circ Physiol 279: H1383-H1391, 2000;
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Vol. 279, Issue 3, H1383-H1391, September 2000

Polyamines decrease Ca2+ sensitivity of tension and increase rates of activation in skinned cardiac myocytes

Samantha P. Harris1, Jitandrakumar R. Patel1, Laurence J. Marton2,3,4, and Richard L. Moss1

Departments of 1 Physiology, 2 Pathology and Laboratory Medicine, and 3 Oncology, University of Wisconsin Medical School, Madison, 53706; and 4 SLIL Biomedical Corporation, Madison, Wisconsin 53711


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Owing in part to their interactions with membrane proteins, polyamines (e.g., spermine, spermidine, and putrescine) have been identified as potential modulators of membrane excitability and Ca2+ homeostasis in cardiac myocytes. To investigate whether polyamines also affect cardiac myofilament proteins, we assessed the effects of polyamines on contractility using rat myocytes and trabeculae that had been permeabilized with Triton X-100. Spermine, spermidine, and putrescine reversibly increased the [Ca2+] required for half-maximal tension (i.e., right-shifted tension pCa curves), with the following order of efficacy: spermine (+4) > spermidine (+3) > putrescine (+2). However, synthetic analogs that differed from spermine in charge distribution were not as effective as spermine in altering isometric tension. None of the polyamines had a significant effect on maximal tension, except at high concentrations. After flash photolysis of DM-Nitrophen (a caged Ca2+ chelator), spermine accelerated the rate of tension development at low and intermediate but not high [Ca2+]. These results indicate that polyamines, especially spermine, interact with myofilament proteins to reduce apparent Ca2+ binding affinity and speed cross-bridge cycling kinetics at submaximal [Ca2+].

spermine; cardiac muscle; myofilaments


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

POLYAMINES SUCH AS PUTRESCINE, spermidine, and spermine are biological cations that are present in all mammalian cells. In these cells, polyamines are an absolute requirement for normal cell growth and division, and abnormal polyamine expression is associated with tumorigenesis, altered gene expression, and induction of apoptotic pathways (11). In the heart, aspects of both developmental and hypertrophic growth are associated with changes in polyamine metabolism, and it is known that induction of a rate-limiting enzyme in their synthesis, ornithine decarboxylase (ODC), is among the first responses to numerous hormonal and trophic stimuli (for review, see Ref. 18). In some instances, such as hypertrophy induced by beta -adrenergic agonists, inhibition of ODC attenuates cardiac growth (2, 12).

Although the importance of polyamines to cell cycle and growth regulation has been recognized for some time, the exact mechanisms by which polyamines exert their effects are not yet fully understood. Owing in large part to their positive charge at neutral pH, the ability of polyamines to bind to and stabilize nucleic acids and proteins is thought to be essential for aspects of DNA, RNA, and protein function (11). In addition, polyamines are known to bind with varying affinity to a variety of cytoplasmic ligands including many membrane proteins. The potential for these interactions to affect cell cycle regulation has been suggested (44), as have hypotheses that polyamines play more direct functional roles as cytoplasmic modulators in various cell processes.

For cardiac and other excitable cell types, the hypothesis that polyamines function as intracellular modulators has been strengthened by recent findings that polyamines, especially spermine, bind with high affinity to several types of ion channels and affect their function (for review, see Ref. 47). For instance, binding of polyamines to many types of inward rectifier potassium channels (Kir channels) results in a blocked channel state that gives rise to the anomalous conductance properties of these channels (26, 25). Furthermore, because Kir channels are the dominant means of conductance at resting membrane potentials in myocardium, modulation of their gating properties by changes in cytoplasmic polyamines has been suggested as a mechanism whereby cell excitability and action potential shape can be regulated (8, 30, 36).

Cell contractility may also be affected by polyamines. For example, in isolated myocytes (45), strips of ventricular myocardium (5) and smooth muscle (31) exogenous polyamines reduce cell contractility, presumably by blocking Ca2+ channels and reducing intracellular [Ca2+]. However, intracellular polyamines may have opposite effects on cell contractility. For instance, in smooth muscle that had been permeabilized to permit access to the cytosolic compartment, polyamines potentiated contractile responses by increasing myofilament Ca2+ sensitivity of tension (31, 39). These potentiating effects might be specific to smooth muscle, however, because spermine and spermidine reduced MgATPase activity and tension in glycerol-treated skeletal muscle (13, 14), and the effects of cytoplasmic polyamines on cardiac myofilaments have not yet been investigated.

Therefore, in light of their potential to modulate membrane excitability and contractility, the present study was undertaken to investigate the effects of polyamines on cardiac myofilament proteins. Detergent permeabilized ("skinned") myocytes and trabeculae were used to allow access of polyamines to myofilaments and to minimize potential interactions with membrane proteins and consequent effects on intracellular Ca2+ handling. Results of the study show that polyamines, particularly spermine, reduce myofilament Ca2+ sensitivity of tension and speed the kinetics of tension development through direct interactions with myofilament proteins. The results are consistent with a role for cytoplasmic polyamines in contributing to the overall contractile properties of cardiac muscle.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals and solutions. Chemicals, including HCl salts of putrescine, spermidine, and spermine, were purchased from Sigma Chemical (St. Louis, MO), except CaCl2 (Orion Research), propionic acid (Fluka), and DM-Nitrophen (Calbiochem). Synthetic polyamine analogs were generously provided by Benjamin Frydman of SLIL Biomedical (Madison, WI).

Relaxing and Ca2+-activating solutions for measurements of Ca2+ sensitivity of force contained (in mmol/l) 20 imidazole, 7 EGTA, 4 MgATP, 5.4 MgCl2 (1 free Mg2+), 14.5 creatine phosphate, and sufficient KCl to adjust ionic strength to 180 mmol/l. In addition, relaxing and maximal activating solutions contained CaCl2, such that free Ca2+ concentration ([Ca2+]free) was pCa 9.0 and 4.5, respectively, where pCa = -log [Ca2+]. Solution pH was titrated with KOH to 7.0 at 15°C. A computer program (15) and published stability constants (corrected for pH and temperature) (20) were used to calculate the final concentrations of each metal, ligand, and metal-ligand complex in solution. Solutions containing a range of [Ca2+]free (i.e., pCa 6.2-5.2) were prepared by mixing solutions of pCa 9.0 and pCa 4.5.

For measurement of rates of tension development, relaxing (pCa 9.0) and maximal activating (pCa 4.5) solutions were similar to those described above except that 1) 100 mmol/l N, N-bis[2-hydroxyethyl]-2-aminoethane sulfonic acid (BES) was used in place of imidazole; 2) ionic strength was adjusted with potassium propionate; and 3) 5 mmol/l dithiothreitol (DTT) was added to each solution. The preactivating solution contained (in mmol/l) 0.07 EGTA, 79 potassium propionate, 4.77 ATP, 5.29 MgCl2, 100 BES, 5 DTT, and 15 creatine phosphate. Loading solution contained (in mmol/l) 1 DM-Nitrophen, 0.4 CaCl2, 75.2 potassium propionate, 4.77 ATP, 5.96 MgCl2, and 15 creatine phosphate. The apparent stability constants for Ca2+-EGTA and Ca2+-DM-Nitrophen were 2.39 × 106 M-1 and 2.0 × 108 M-1, respectively (22).

Preparation of ventricular myocytes and trabeculae. Adult female Sprague-Dawley rats were anesthetized by inhalation of methoxyflurane and their hearts were rapidly excised. Upon excision, hearts were cannulated and mounted on a modified Langendorff apparatus for retrograde perfusion via the coronary circulation. Ventricular myocytes were obtained by enzymatic digestion of hearts as described previously (37). Myocytes were skinned by incubation for 6 min at 22°C in relaxing solution containing (in mmol/l) 1.0 free Mg2+, 100 KCl, 2.0 EGTA, 4.0 ATP, and 10 imidazole; and 0.03% Triton X-100. Skinned myocytes were washed twice in detergent-free relaxing solution and stored on ice for use the same day. For isolation of trabeculae, hearts were initially perfused with an ice-cold cardioplegic solution containing (in mmol/l) 140 NaCl, 15 KCl, 1.2 MgCl2, 2.0 NaH2PO4, 5.0 sodium acetate, 10 HEPES, and 10 glucose, until perfusate exiting the heart was visibly clear of blood. Hearts were then dissected and strips (130-210 µm wide) of unbranched trabeculae, running between the right ventricular free wall and tricuspid valve, were removed and skinned for 30 min in a solution containing (in mmol/l) 58.2 potassium proprionate, 7.0 EGTA, 4.75 ATP, 5.45 MgCl2, 0.02 CaCl2, 100 BES at pH 7.0, and 5 DTT; and 1% Triton X-100.

Ca2+ sensitivity of tension. Ca2+ sensitivity of tension in skinned single myocytes was determined as described previously (37). Myocytes were placed in relaxing solution on the stage of an inverted microscope (Zeiss), and a single myocyte was attached with silicone adhesive (Dow Corning) to two stainless steel pins. One pin was attached to the active element of a force transducer (model 403, Cambridge Technology) and the other to a length controller. The length controller was a piezoelectric translator (Physik Instrumente) or, in some cases, a torque motor (model 308B, Cambridge Technology). Both the force transducer and the length controller were mounted on three-way micromanipulators (Narishige) anchored to a vibration isolation table.

After the silicone adhesive was allowed to cure (~45 min), myocytes were raised up off the stage and transferred to solution of pCa 9.0. Sarcomere length was adjusted to 2.3 µm using online video imaging. Isometric tension was measured at 15°C by transferring the myocyte to activating solutions containing a range of [Ca2+]free and measuring the difference in force just before and after a rapid slack step (~20% of cell length). Ca2+-activated tension was determined as the difference between isometric tension generated in activating solution (pCa 6.1-4.5) and in relaxing solution (pCa 9.0). Isometric forces (P) at submaximal pCa were expressed as a fraction of maximal force (Po) measured at pCa 4.5, such that relative force (PRel) = P/Po.

Effects of polyamines on tension were assessed after an initial 15-min incubation of the myocyte in relaxing solution containing the desired concentration of polyamine. Force measurements were then repeated with polyamines in all activating and relaxing solutions. To assess reversibility of polyamine effects, measurements were repeated following three solution changes over a 10-min period using fresh relaxing solution that lacked polyamines.

Rate of tension development. The kinetics of force development were measured by recording the increase in tension following flash photolysis of caged Ca2+ from the chelator, DM-Nitrophen. Skinned trabeculae were placed in a stainless steel experimental chamber and attached at their ends to the arms of a length controller (model 350, Cambridge Technology) and force transducer (model 403, Cambridge Technology), as previously described by Moss et al. (28). The chamber assembly was then placed on the stage of an inverted microscope (Olympus) fitted with a charge-coupled device camera. Light from a halogen lamp was passed through a cutoff filter (lambda  > 620 nm) and then used to illuminate the preparation for video image analysis. Sarcomere length was maintained at ~2.35 µm throughout the course of an experiment.

Po measurements were performed by transferring each trabecula from the relaxing solution to preactivating solution (2 min) and then to a maximally activating solution (pCa 4.5). Once tension reached a steady level, the preparation was rapidly slackened and returned to relaxing solution. For measurements of the rate of tension development, trabeculae were transferred from a preactivating solution to a loading solution containing 1 mmol/l DM-Nitrophen and CaCl2. At the end of a 5-min incubation in loading solution, the trabeculae were transferred to an 80-µl quartz-walled photolysis chamber filled with silicone oil (Dow Corning 200 fluid, viscosity 10 cs). Rapid release of Ca2+ from DM-Nitrophen was achieved using a flash of ultraviolet (UV) light (lambda  = ~360 nm) from a xenon lamp (Optoeletronik). After measurement of the postflash active force (P), preparations were returned back to the relaxing solution. Three cycles of loading and photolysis were randomly performed to achieve low, intermediate, and high levels of postflash force by photolyzing DM-Nitrophen with three different intensities of UV light (generated by adjustment of the power supply to the UV flash lamp). After measurements of postflash tension with DM-Nitrophen, maximum force at pCa 4.5 was again determined to assess rundown of the preparation.

To examine the effects of spermine on the rate of force development, preparations were incubated in the relaxing solution containing 1 mmol/l spermine for 15 min before repeating the above protocol. All solutions, except maximum activating solutions, contained 1 mmol/l spermine. Tension generated by each trabecula in the presence of spermine was expressed relative to maximum tension (in pCa 4.5) recorded immediately after the 15-min incubation in relaxing solution containing spermine.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of spermine on Ca2+ sensitivity of tension. The Ca2+ sensitivity of tension of single ventricular myocytes was determined by measuring developed isometric tension as a function of activating [Ca2+]. Figure 1 shows a comparison of normalized tension-pCa relationships obtained from a single myocyte before (control) and after inclusion of 1 mmol/l spermine in relaxing and activating solutions. In the presence of spermine, the tension-pCa relationship was shifted to the right relative to control and the pCa at which force was half-maximal (pCa50) was decreased. Summary data from five experiments (Table 1) showed that spermine significantly reduced pCa50 values, indicating a reduction in the apparent Ca2+ binding affinity of the myofilament proteins by spermine. The steepness of the tension-pCa curves was also somewhat increased in the presence of spermine as indicated by an increase in Hill coefficients (nH) (Table 1). Although the latter suggests that the cooperativity of tension activation may be enhanced by spermine, the result should be regarded as tentative due to the lack of robustness of the test statistics (power = 0.59). The effects of spermine were readily reversed following a brief (10-15 min) washout period and elimination of spermine from activating and relaxing solutions.


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  		Fig. 1.   Effect of spermine (SPM) on Ca2+ sensitivity of isometric tension. Tension-pCa relationships obtained from a single skinned myocyte were determined first in absence of SPM and then after 15-min incubation of the myocyte in relaxing solution containing 1 mmol/l SPM. For tension measurements in presence of SPM, 1 mmol/l SPM was present in all activating and relaxing solutions. Tension-pCa relationships were determined again after 15-min washout of SPM from all solutions. Steady-state force (P) was expressed relative to maximum steady-state force (Po) in solution of pCa 4.5. Smooth lines were fit data in presence and absence of SPM using the Hill equation: P<IT>/</IT>P<SUB>o</SUB><IT>=</IT>[Ca<SUP>2+</SUP>]<SUP><IT>n</IT><SUB>H</SUB></SUP><IT>/</IT>(<IT>k</IT><SUP><IT>n</IT><SUB>H</SUB></SUP><IT>+</IT>[Ca<SUP>2+</SUP>]<SUP><IT>n</IT><SUB>H</SUB></SUP>)<IT>,</IT> where nH is the Hill coefficient and k denotes the pCa at which relative force is half-maximal, i.e., pCa50. In absence of SPM, pCa50 was 5.67 and was reduced to 5.49 in presence of 1 mmol/l SPM. pCa50 after washout of SPM was 5.66.


                              
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  		Table 1.   Summary of tension-pCa curve fits in the absence and presence and after washout of 1 mmol/l spermine

To investigate the concentration dependence of the shift in midpoint of the tension-pCa relationship and to compare the effects of spermine, spermidine, and putrescine on submaximal tension, the effects of increasing polyamine concentration were assessed at a single submaximal activating [Ca2+]. For each experiment, the pCa of the activating solution was selected so that tension in the absence of polyamines was close to 50% of maximum Ca2+-activated tension, i.e., near the pCa50 for each myocyte. Figure 2A shows that addition of spermine and spermidine to activating and relaxing solutions reduced submaximal Ca2+-activated tension in a dose-dependent manner. Spermine was more effective than spermidine, with significant reductions in tension apparent at concentrations >= 400 µM. Putrescine was the least effective, and reductions in tension were minimal even at the highest concentration tested (6.4 mmol/l). Although low putrescine concentrations (e.g., 200 µM) appeared to potentiate tension, the effects were not statistically significant (P > 0.05). Effects of polyamines on resting tension in relaxing solutions (pCa 9.0) were not observed.


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  		Fig. 2.   Effects of polyamines on submaximal tension in skinned myocytes. For each experiment, an activating solution of pCa 5.6-5.8 was chosen to produce relative submaximal tension close to 50% of maximum Ca2+-activated tension (i.e., near pCa50). Increasing concentrations of polyamines were then added to relaxing and activating solutions and ~3 min after each addition tension was again measured. After the tension measurement at the highest polyamine concentration shown, myocytes were incubated in fresh relaxing and activating solutions (lacking polyamines) and tension was again measured (washout). Symbols represent the means ± SE of four or more experiments. A: effects of SPM, spermidine, and putrescine on submaximal tension. B: effects of bis(ethyl) synthetic polyamine analogs (BE-3-4-3 and BE-4-4-4-4) on submaximal tension. SPM data as in A were replotted for comparison. C: effects of SPM in pCa solutions containing 1 mmol/l free Mg2+ and either 4 mmol/l or 2 mmol/l MgATP.

At neutral pH, polyamines are protonated and carry net positive charge. Spermine bears a net +4 valence, and spermidine and putrescine carry +3 and +2 charges, respectively. Because the relative efficacy of polyamines at reducing tension was correlated with polyamine valence (+4 > +3 > +2), the possibility that the effects of spermine on tension were due primarily to counter-ion effects was investigated by comparing the ability of synthetic polyamine analogs to inhibit submaximal Ca2+-activated tension. As shown in Fig. 2B, the bis-ethylated polyamine analogs, N1,N12-bisethylspermine (BE-3-4-3) (7) and 1,19,-bis-(ethylamino)-5,10,15-triazanondecane (BE-4-4-4-4) (4) bearing net +4 and +5 charges, respectively, were less effective than spermine at reducing tension. The analogs differ from spermine in that they lack primary amine groups and hence differ in their charge distribution. Therefore, the ability of spermine to reduce Ca2+ sensitivity of tension does not appear to depend entirely on the total charge of the polyamine, and other structural properties are also likely to be important. Consistent with this, polyamine charge distribution and overall length have been shown to be relevant factors in determining the specificity of polyamine interactions (3, 27).

Polyamines may also compete with Mg2+ for binding at protein divalent cation binding sites and nucleotide phosphate moieties (29). Because all solutions used here contained 1 mmol/l calculated free Mg2+ and 4 mmol/l MgATP, experiments were done to test the possibility that the effects of spermine to reduce submaximal tension depend on concentrations of these constituents. Figure 2C shows the effects of spermine on myocte tension in solutions containing 1 mmol/l free Mg2+ and 2 mmol/l MgATP. When [MgATP] was reduced, the effect of spermine to inhibit tension was increased relative to its effects in solutions containing 4 mmol/l MgATP. This result is consistent with spermine binding to ATP phosphate moieties and suggests that polyamines added to myocyte bath solutions are effectively buffered by ATP. Thus the polyamine concentration required to elicit contractile effects is likely to be an overestimate relative to the concentration required in ATP-free solutions. Also, the finding that spermine had greater effects when MgATP was reduced suggests that a secondary increase in Mg2+ is not the primary mechanism by which spermine reduces tension.

Effects of spermine on maximum Ca2+-activated tension. To determine whether polyamine-induced reductions in tension were related to decreases in the force-generating capabilities of myosin cross bridges (e.g., the number of cross bridges or the force per cross bridge), effects of spermine on maximum Ca2+-activated tension (Po) were investigated. A single concentration of spermine was tested in each myocyte by first activating in a pCa 4.5 solution in the absence of spermine and again after the addition of spermine to relaxing and activating solutions. A final tension measurement was made following washout of spermine from all solutions. As shown in Fig. 3, spermine significantly reduced maximum tension at concentrations >= 3.2 mmol/l. Effects of spermine were reversed upon removal of spermine from relaxing and activating solutions.


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  		Fig. 3.   Effects of SPM on maximum Ca2+-activated tension. Maximum Ca2+-activated tension (pCa 4.5) was measured in absence and presence of SPM and again after 15-min washout of SPM from solutions. Tension in presence of SPM and after washout was expressed relative to initial maximum tension measurement in absence of SPM. Bars represent means ± SE of three or more experiments. *Significantly different from control (0 mmol/l SPM). P < 0.05, one-way ANOVA.

Figure 4 shows cumulative effects of spermine, spermidine, and putrescine on maximum tension. Similar to effects on submaximal tension, the order of efficacy for reducing maximum tension was spermine > spermidine > putrescine. However, in contrast to effects on submaximal tension, reductions in tension by spermine (+4) at saturating [Ca2+] were not different from those of the synthetic spermine analog BE 3-4-3 (+4) (data not shown). These data suggest that at maximal activation, the effects of spermine to inhibit tension are relatively nonspecific, related more to total polyamine valence than to other structural characteristics.


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  		Fig. 4.   Cumulative effects of polyamines on maximum Ca2+-activated tension. For each experiment, maximum Ca2+-activated isometric tension was initially measured in solution of pCa 4.5. Increasing concentrations of polyamines were then added to relaxing (pCa 9.0) and activating solutions and tension measurements were repeated. After the tension measurement at the highest polyamine concentration shown, myocytes were incubated in fresh relaxing and activating solutions (without polyamines) and tension was again measured (washout). Symbols represent means ± SE of four or more experiments. SPD, spermidine; PUT, putrescine.

Effects of spermine on kinetics of tension development. Alterations in myofilament Ca2+ sensitivity of tension have been related to changes in one or more of three variables: 1) the Ca2+ binding affinity of the thin filament protein troponin C (TnC); 2) cooperative interactions among myofilament proteins; and 3) the rate of cross-bridge cycling. Because the slope of the pCa-tension relationship, which provides an index of apparent cooperativity among the contractile proteins, was only modestly affected by spermine (Table 1), it seemed unlikely that reductions in Ca2+ sensitivity were due primarily to changes in cooperative activation by spermine. To distinguish between the remaining possibilities, we measured the rate constant of tension development (kCa) following rapid release of Ca2+ from a photolabile Ca2+ chelator, DM-Nitrophen (32). This measurement is analogous to the rate constant of tension redevelopment (ktr) following a release and restretch maneuver and has been interpreted as a net rate constant describing cross-bridge cycling kinetics (9).

The effects of 1 mmol/l spermine on the tension development in a skinned trabecula following flash photolysis of DM-Nitrophen are shown in Fig. 5. Before photolysis of DM-Nitrophen, trabeculae generated no measurable tension in a loading solution containing 0.4 mmol/l Ca2+ buffered with 1 mmol/l DM-Nitrophen (calculated free Ca2+, pCa 5.85). After flash photolysis of DM-Nitrophen, trabeculae developed tension that was proportional to the intensity of the UV flash. Under control conditions (Fig. 5A), photolysis with low, intermediate, and high levels of UV light resulted in steady isometric tensions of 0.41, 0.70, and 0.92 Po. Figure 5B shows the same data normalized to the peak tension after flash. The half-times of tension development were 146, 114, and 76 ms, respectively. These data indicate that the rate of tension development increased with [Ca2+], in agreement with previous reports of the Ca2+ dependence of the rate of force development in intact (1) and skinned myocardium (17, 48).


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  		Fig. 5.   Effects of SPM on isometric tension and rate of tension development in skinned cardiac trabeculae. A: skinned trabeculae were incubated in loading solution (0.4 mmol/l CaCl2 and 1 mmol/l DM-Nitrophen, calculated free [Ca2+] = pCa 5.85) for 3 min, transferred to silicone oil in a quartz trough, and then exposed to a low- (c, 150 V), intermediate- (b, 200 V), or high- (a, 350 V) intensity ultraviolet (UV) light flash (F) to achieve variable release of Ca2+. Tension was recorded and trabeculae were then returned to relaxing solution. Three separate tension traces from the same trabecula are shown and are expressed relative to tension generated in solution of pCa 4.5. B: data in A were normalized to the peak tension generated at each flash intensity (a, b, and c). C and D: tension traces recorded from the same trabecula in the presence of 1 mmol/l SPM. The trabecula was incubated for 15 min in a relaxing solution containing 1 mmol/l SPM, and tension measurements were done as described in A. All solutions, except pCa 4.5, contained 1 mmol/l SPM.

Similar measurements repeated in the presence of 1 mmol/l spermine resulted in steady tensions of 0.34, 0.56, and 0.77 Po in response to the same low-, intermediate-, and high-intensity UV flashes (Fig. 5C). Thus consistent with the steady-state measurements in single skinned myocytes, 1 mmol/l spermine reduced tension at each submaximal [Ca2+]. However, as shown in Fig. 5D, the half-times to peak tension following low- and intermediate-intensity but not high-intensity flashes were decreased in the presence of spermine (116, 104, and 74 ms, respectively).

Summary data obtained in the absence and presence of 1 mmol/l spermine are shown for five trabeculae in Fig. 6. Half-time to peak tension is plotted versus tension amplitude, expressed relative to maximum tension (Po) in the same preparations, following photolysis with low-, intermediate-, and high-intensity flashes. Comparison of the two curves indicates that spermine shifted the Ca2+ dependence of the activation kinetics such that rate of tension development was sped (decreased half-time to peak tension) at low and intermediate levels of Ca2+. At high-flash intensity (high [Ca2+]), rates were similar in the presence and absence of spermine. Thus spermine alters the Ca2+ dependence of the tension development rate without affecting the maximum rate of tension development.


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  		Fig. 6.   Summary data of the effects of SPM on tension and rate constant of tension development in skinned cardiac trabeculae. Relative tension following flash photolysis of DM-Nitrophen with low, intermediate, and high intensities of UV light were plotted against half-time to peak tension. The means ± SE of 5 trabeculae in the absence (open circle ) and presence () of 1 mmol/l SPM are shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The primary determinants of the force and speed of contraction in intact myocardium are the amplitude and duration of the intracellular Ca2+ transient, the responsiveness of the myofilaments to Ca2+, and the intrinsic cycling kinetics of the contractile proteins. Previous studies suggested that cell excitability (23, 25) and Ca2+ delivery (45) might be influenced by polyamines. Results of the present study, done with detergent-permeabilized myocytes to allow control of [Ca2+] in solutions bathing the myofilaments, provide the first evidence that polyamines can affect cardiac contractility through direct effects on myofilament proteins. In the presence of polyamines, the Ca2+ sensitivity of tension is decreased and cross-bridge cycling kinetics are increased.

Mechanisms of polyamine effects. A decrease in the apparent Ca2+ binding affinity of the myofilaments could result from a direct competitive effect of spermine to reduce Ca2+ binding by the thin-filament regulatory protein TnC. Because Ca2+ binding by TnC was not directly measured in the present study, this possibility cannot be eliminated. However, observations that spermine produced differential effects on tension and the rate constants of tension development (kCa) suggest that other mechanisms in addition to or in place of effects on TnC are involved. For example, in skinned skeletal muscle fibers calmidazolium increases TnC-Ca2+ binding affinity resulting in increases of both isometric force and the rate of tension development (i.e., ktr) (34). Therefore, it seems unlikely that a reduction in TnC-Ca2+ binding by spermine could itself account for both the decreased Ca2+ sensitivity of tension and increased rates of tension development.

On the other hand, the effect of spermine to increase kCa at submaximal [Ca2+] is indicative of increased cross-bridge cycling kinetics. Because binding of Ca2+ to TnC is thought to be rapid relative to the rates of cross-bridge attachment and detachment, measurements of the rate of tension development (e.g., kCa and ktr) have previously been interpreted in terms of the apparent rate constants limiting cross-bridge transitions to and from attached, force-generating states (9). According to a two-state model, with one attached and one detached state (e.g., 9), kCa is equivalent to the sum of the apparent forward (fapp) and reverse (gapp) rate constants. Thus the current observations that kCa is increased at submaximal Ca2+ is compatible with effects of spermine to either increase the rate of cross-bridge attachment or the rate of detachment. However, because steady-state force is proportional to fapp/(fapp + gapp), an increase in gapp, the rate of cross-bridge detachment, most easily accounts for the simultaneous decrease in force and increase in rate of tension development. Further experiments are necessary to test this hypothesis.

Other explanations for the differential effects of polyamines on tension and rate of tension development are also possible. For instance, kinetic schemes that incorporate additional cross-bridge states (35) or that include cooperative interactions that act to promote thin filament activation (10) may be necessary to describe the dual effects of spermine on steady-state tension and rate of tension development. For example, additional strongly bound, non-force-generating cross-bridge states were used to describe the opposing effects of caffeine (46) and Pi (35) on tension and cross-bridge cycling kinetics. Of potential relevance, spermine is known to bind phosphate moieties with high affinity (29). By stabilizing a phosphate-bound, cross-bridge state, spermine could potentially influence cross-bridge transitions and thereby mimic the ability of Pi to reduce tension and accelerate cycling kinetics.

Muscle-type specificity of polyamine effects. The contractile effects reported here for polyamines in cardiac muscle differ from those in smooth muscle and may reflect tissue-specific differences between muscle types. For instance, at concentrations comparable to those used here, polyamines increase myofilament Ca2+ sensitivity in mildly permeabilized smooth muscle cells (31, 38). However, the effects observed in smooth muscle result primarily from inhibition of myosin phosphatase activity, resulting in increased phosphorylation of myosin regulatory light chains (RLCs) (40). Although not directly assessed in the current study, increased RLC phosphorylation in cardiac cells is also associated with force potentiation (16). Therefore, it seems unlikely that the effects of polyamines in heart cells are mediated through analogous mechanisms. Furthermore, the more extensive permeabilization of the cardiac myocytes with Triton X-100 and the ability to rapidly wash out the effects of polyamines argues against changes in the phosphorylation state of myofilament proteins as a primary mechanism by which the polyamine effects are mediated. Thus the effects of polyamines observed in the current study apparently occur independent of changes in phosphorylation state and are due instead to direct interactions with myofilament proteins.

Direct stimulatory effects of polyamines have been observed in smooth muscle in the absence of changes in RLC phosphorylation (38, 42). The mechanism of these effects is potentially related to the ability of poly-L-lysine, a polycationic peptide, to promote the transition of myosin from a globular (10S) conformation to a filamentous (6S) form (41). Because myosin from striated muscle does not undergo such conformational changes upon activation, these effects may also be specific to smooth muscle. However, because the molecular basis for direct polyamine effects have not been identified for either smooth or cardiac muscle, it is possible that the sites of interaction are similar.

In contrast to smooth muscle, the effects of polyamines in cardiac cells are similar to effects observed in skeletal muscle. For instance, in glycerin-treated skeletal muscle, polyamines reduced tension and MgATPase activity (13, 14). In addition, in skinned psoas fibers, steady-state tension was reduced and activation kinetics at submaximal [Ca2+] were sped by 1 mmol/l spermine (S. P. Harris, J. R. Patel, L. J. Marton, and R. L. Moss, unpublished observations). These data suggest that the contractile effects of polyamines are similar in cardiac and fast skeletal fibers and therefore may be common to all striated muscle.

Physiological implications. On the basis of observations that the Ca2+ sensitivity of tension differs between intact and skinned cardiac trabeculae, Gao et al. (19) proposed the existence of soluble Ca2+ sensitizers that contribute to the inotropic properties of cardiac muscle. Although the effects of polyamines to reduce steady-state Ca2+ sensitivity in skinned myocytes would seem to preclude a role in Ca2+ sensitization, the effects of spermine reported here occur at concentrations compatible with values of total polyamine content in heart and so may influence overall contractile status. For example, a survey of mouse striated muscles showed that polyamine concentrations were related to muscle type and were highest in cardiac muscle (24). Among the different polyamines, the concentration of spermine was greatest, i.e., 440 nmol/g, a value in close agreement to frequently reported values for polyamine content of rat heart (200-400 nmol/g) (21, 33).

Moreover, polyamine synthetic enzymes are highly inducible and the cellular content of all three polyamines can be elevated severalfold in response to a variety of inotropic stimuli, including beta -adrenergic agonists (18). Such changes in polyamine content could potentially affect cell contractility through interactions with myofilament proteins as described here. For example, the effects of beta -adrenergic stimulation are qualitatively similar to those induced by spermine (37). Polyamine interactions with myofilament proteins may thereby contribute to or modulate inotropic responsiveness. Consistent with this, metabolic inhibition of polyamine synthesis in rats produced reductions of cardiac putrescine and spermidine levels that were associated with decreased inotropic responsiveness to ouabain, norepinephrine, and Ca2+ in vitro (6). Thus despite uncertainties regarding the concentrations of free cytoplasmic polyamines and their subcellular distributions (11, 43), basal or stimulated cardiac inotropic state may be affected through polyamine interactions with myofilament proteins as described here.


    ACKNOWLEDGEMENTS

The authors thank Dr. Benjamin Frydman for the generous gift of synthetic polyamine analogs.


    FOOTNOTES

This work was supported by National Institutes of Health Grant 47053 (to R. L. Moss) and was done during the tenure of a postdoctoral fellowship (to S. P. Harris) from the American Heart Association, Northland Affiliate.

Address for reprint requests and other correspondence: S. P. Harris, 109 SMI, 1300 University Ave., Madison, WI 53706 (E-mail: spharris{at}facstaff.wisc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2 December 1999; accepted in final form 15 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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