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Cardiovascular Research 1998 40(3):483-491; doi:10.1016/S0008-6363(98)00201-6
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Ryanodine and the left ventricular force–interval and relaxation–interval relations in closed-chest dogs: insights on calcium handling

Sumanth D. Prabhua,b,*

aDepartment of Medicine/Cardiology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San AntonioTX 78284-7872, USA
bAudie Murphy Memorial Veterans Hospital, San Antonio TX 78284, USA

* Tel.: +1-(210)-567-4600; fax: +1-(210)-567-6960; e-mail: Prabhu@uthscsa.edu

Received 19 January 1998; accepted 23 April 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Although the myocardial force–interval and relaxation–interval relations are considered to be mechanical expressions of myocardial Ca2+ handling, correlation of these phenomena with altered Ca2+ kinetics in the intact state is limited. Thus, I sought to determine the impact of selective impairment of physiologic sarcoplasmic reticulum Ca2+ release, achieved by the use of the drug ryanodine, on these relations in the intact animal. Methods: Twelve dogs instrumented with left ventricular manometers and piezoelectric dimension crystals were studied before and after ryanodine (4 µg/kg intravenously). End-systolic elastance was measured at paced heart rates of 120–180 bpm to determine the force-frequency response. Mechanical restitution and relaxation restitution were determined by measuring contractile (single beat elastance) and relaxation (peak negative dP/dt) responses for beats delivered at graded extrasystolic intervals, with normalized responses expressed as a function of extrasystolic interval. Results: Ryanodine accelerated mechanical restitution (time constant 60.3±3.9 versus 81.7±10.1 ms, p<0.05) and reduced maximal contractile response (107.5±2.1 versus 122.1±5.7%, p<0.05), slowed early relaxation restitution (time constant 65.5±13.8 versus 36.8±3.8 ms, p<0.05) without changing late relaxation restitution kinetics, and amplified the force-frequency response (end-systolic elastance, 180 bpm, 19.4±4.3 versus 11.4±1.2 mm Hg/ml, p<0.05). Conclusions: These findings suggest that in the intact animal, Ca2+ handling by the sarcoplasmic reticulum is a primary determinant of mechanical restitution and early relaxation restitution, but not late relaxation restitution. Conversely, ryanodine induced augmentation of the force-frequency response indicates a central role for sarcolemmal Ca2+ influx in producing frequency potentiation.

KEYWORDS MR, Mechanical restitution; FFR, Force–frequency response; RR, Relaxation restitution; SR, Sarcoplasmic reticulum; SL, Sarcolemma; LV, Left ventricle, left ventricular; P, Pressure; V, Volume; dAP, Anterior–posterior diameter; dSL, Septal–lateral diameter; dLA, Long axis diameter; ECG, Electrocardiogram; PES–VES, End-systolic pressure–volume; HR, Heart rate; ESI, Extrasystolic interval; VCF, Mean velocity of circumferential fiber shortening; VLV, Left ventricular volume; ED, End-diastole, end-diastolic; ES, End-systole, end-systolic; EES, End-systolic elastance; V0, Volume intercept of end-systolic pressure–volume relation; SW, Stroke work; SBE, Single beat elastance; CRmax, Maximal contractile response; TC, Time constant; ESI0, ESI axis intercept of mechanical restitution curve; TCM, Time constant of mechanical restitution; TCR, Time constant of relaxation restitution; RRmax, Asymptotic plateau of relaxation response


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The effect of temporary changes in stimulation pattern on the strength of cardiac contraction is described by the force–interval relation. Two aspects of this relation, mechanical restitution (MR) and the force-frequency response (FFR), refer to the monoexponential, time-dependent restoration of contractile function following a depolarization, and the augmentation of contractility resulting from increases in stimulation frequency, respectively. Prior studies have shown that MR and FFR are mechanistic expressions of Ca2+ handling by both the sarcoplasmic reticulum (SR) and sarcolemma (SL) [1, 2], and as such can be considered macroscopic measures of excitation–contraction coupling. Importantly, the correlation of force–interval behavior to intracellular Ca2+ handling has been investigated predominantly in isolated muscle preparations [3–6], with very little comparable data from the intact state. Such studies would be desirable since there are important quantitative differences in intact animals, including significantly faster MR kinetics [7], and variability in the expression of the FFR depending on the prevailing experimental conditions [8–10].

Prabhu and Freeman [11]have shown that, analogous to the recovery of contractile force, cardiac relaxation also follows a pattern of restitution. Relaxation restitution (RR) is biphasic and can be described by two concatenated monoexponential curves. The first phase parallels the MR curve, with relaxation recovering more rapidly than contractile force. The second phase consists of gradual slowing of relaxation at long cycle lengths. The relationship between RR and intracellular Ca2+ handling has not been rigorously evaluated. Since early RR kinetics can be predicted by recirculating models of SR Ca2+ handling put forth by several investigators to explain MR behavior [3, 12], we previously reasoned that early RR may also be an expression of SR Ca2+ handling. Late RR is not predicted by these models and the underlying mechanisms of this phase are less clear.

Accordingly, the purpose of the current investigation was to determine the impact of selective impairment of physiologic SR Ca2+ release, achieved through the use of the drug ryanodine, on the force–interval and relaxation–interval relations in the intact animal. My objective was threefold: (1) to confirm the dependence of MR on SR Ca2+ release in the intact animal and corroborate findings from isolated muscle studies, (2) to define the relationship between RR and SR Ca2+ handling and (3) to define the link between the FFR and intracellular Ca2+ dynamics in the intact state.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Experimental protocol
All animal studies were performed in accordance with guidelines described in the NIH Guide for the Care and Use of Laboratory Animals (DHHS publication No. [NIH] 85-23, revised 1985). Under general anesthesia, twelve mongrel dogs of either sex were surgically instrumented for long term physiologic monitoring as previously described [11, 13]. The instrumentation included a high fidelity micromanometer (Konigsberg Instruments) implanted across the left ventricular (LV) apex, three sets of piezoelectric crystals implanted in the LV endocardium along the anterior–posterior (dAP), septal–lateral (dSL), and long axis (dLA) diameters, balloon occluder cuffs placed around the inferior vena cava, and pacemaker wires sutured to the left atrium. The animals recovered a minimum of 2 weeks prior to experimentation.

All experiments were performed with the animal lying in a sling on its right side, and after anesthesia with thiopental sodium (25–30 mg/kg), droperidol (1.5–3.0 mg/kg), and fentanyl (0.03–0.06 mg/kg). Respiration was supported using mechanical ventilation with room air. Autonomic blockade was produced by the administration of intravenous atropine (2 mg) and propranolol (2 mg/kg). All hemodynamic data were collected during 10-s periods of apnea to avoid respiratory effects. Analog tracings of LV pressure (P), dP/dt, the electrocardiogram (ECG), and the three LV dimensions were recorded on a forced ink oscillograph (Beckman Instruments), and simultaneously digitized at a sampling rate of 500 Hz.

Data were collected during steady-state conditions and during rapid caval occlusion to determine the end-systolic pressure–volume (PES–VES) relation. To measure the FFR, steady-state and caval occlusion runs were repeated at heart rates (HR) of 120, 140, 160, and 180 bpm using atrial pacing in ten of the animals. A minimum of 10 min elapsed after each HR change prior to measurement to allow for the development of a new steady-state. Caval occlusion runs which did not display at least a 20 mm Hg drop in peak systolic LVP were discarded. All animals subsequently underwent the cardiac restitution protocol. The atria were paced at a basic cycle length of 375 ms (HR 160 bpm). After an initial series of beats, a single atrial extrastimulus was introduced using a programmable stimulator (Bloom Instruments) over a range of extrasystolic intervals (ESIs). The first ESI was timed to be within the absolute refractory period of the atrioventricular node. The programmed ESI was then increased in 20-ms increments (50 ms increments after ESIs>450 ms) resulting in beats with progressively increasing cycle length. The process was terminated when an intrinsic sinus beat captured the ventricle before the paced beat. At this point, the pacemaker was turned off and a new steady-state was achieved. Intravenous ryanodine was administered at a rate of 0.5 µg/kg/min for a total dose of 4 µg/kg. We have previously shown that this dose results in serum concentrations of ryanodine in the nanomolar range [14]. After stabilization for a period of 30 min, the entire force–interval protocol described above was repeated. After completion of the full experiment, the animals were allowed to recover and were used for other protocols on later dates.

2.2 Data analysis
The digitized data were analyzed using computer software developed in our laboratory. Calculated dP/dt (mm Hg/s) was derived from instantaneous LVP using a running five point Lagrangian fit. The mean velocity of circumferential fiber shortening (VCF, circumferences/s) was defined as the systolic excursion of DAP (mm) divided by the ejection time (s), normalized for end-diastolic diameter [15]. The LV chamber was assumed to be an ellipse [13]and LV volume (VLV, ml) was calculated using the equation: VLV=({pi}/6)·DAP·DSL·DLA. End-diastole (ED) was defined as occurring at the peak of the QRS complex. End-systole (ES) was defined as occurring at the upper left corner of the LV P–V loop. The PES–VES relation was determined using the equation: PES=EES·(VES–V0) by least squares linear regression [16], where EES (mm Hg/ml) is the slope, and V0 (ml) the volume intercept. Stroke work (SW, mm Hg ml) was defined as the area bounded by the P–V loop.

Isovolumic relaxation was defined as occurring between the time of peak negative dP/dt to the time when LVP had fallen to 5 mm Hg above the EDP for that beat [11]. The time constant of LV relaxation, {tau}, was determined by nonlinear regression analysis of the pressure and time data during isovolumic relaxation using the monoexponential function: PLV=(P0–PB)·exp(–t/{tau})+PB, where P0 (mm Hg) is an estimate of LVP at peak negative dP/dt, t is the time (ms), {tau} is the time constant of relaxation (ms), and PB (mm Hg) is the floating pressure asymptote as t approaches infinity [11].

For extrasystolic beats, single beat elastance (SBE) was defined as the maximal ratio of instantaneous LVP to corrected instantaneous VLV (calculated VLV–V0 determined from caval occlusion at the basic cycle length) and used as a measure of contractile response [7, 11]. Extrasystolic relaxation response was assessed using peak negative dP/dt [11], a model-independent parameter. In some animals, due to prolongation of beat duration by ryanodine [14], fusion of the extrasystolic beat with the preceding control beat at small ESIs occurred after ryanodine administration. When this occurred the true extrasystolic response was determined by subtracting the LVP value at the start of the extrasystole from the remainder of the fusion beat. Mechanical and relaxation responses were then calculated for the derived extrasystolic beat as described above.

MR curves were constructed by plotting SBE of the extrasystolic beat, normalized to that of the preceding control beat, against the ESI [7]. RR curves were similarly constructed by plotting the inverse of dP/dtmin for the extrasystolic beat, normalized to that of the preceding control beat, against the ESI [11]. MR and RR curves were fitted to either single or concatenated monoexponential functions using standard nonlinear techniques [7, 11], depending upon whether the curve was monophasic or biphasic. Monoexponential increases in functional responses with time were fitted to the equation: FRN=FRmax ·(1–exp[(ESI0–ESI)/TC]), where FRN is the normalized functional (contraction or relaxation) response (%), FRmax is the maximal value of normalized functional response, ESI0 (ms) is the smallest ESI that produces a response, and TC is the time constant of restitution (ms). Monoexponential decays in functional responses with time were fitted to the equation: FRN=(FR0–FRA)·(exp[(ESI0–ESI)/TC])+FRA, where FR0 is an estimate of FRN at ESI0, and FRa is the plateau asymptote of FRN as ESI approaches infinity.

2.3 Statistical analysis
All group data are expressed as means±S.E.M. Comparisons between mechanical and restitution parameters before and after ryanodine administration were made using the paired t-test. A p value of <0.05 was considered significant. To assess the FFR within experimental groups (i.e. control or ryanodine), comparisons of EES values at each HR to the lowest HR of 120 bpm were made using two-way analysis of variance and paired t-test with Bonferroni correction. Since three comparisons were made under each condition, a p value of <0.0167 was considered significant. To assess the magnitude of FFR between experimental groups, comparisons of EES at each individual HR before and after ryanodine were made using the paired t-test. A p value of <0.05 was considered significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effect of ryanodine on hemodynamic and mechanical parameters
Table 1 lists hemodynamic parameters for the group before and after ryanodine. Ryanodine had no significant effect on HR (p=0.066), LVESP (p=0.29), or LVEDV (p=0.77). However, despite similar HR, preload, and afterload, ryanodine produced marked reductions in dP/dtmax (p<0.001), VCF (p<0.001) and SW (p=0.0017) indicating a significant negative inotropic effect. Additionally, there was a small but significant increase in LVEDP (p=0.024) and significant slowing of LV relaxation with prolongation of {tau} (p<0.001) after ryanodine. Interestingly, ryanodine had no effect on the force-based parameter EES (p=0.34), despite an overall negative inotropic effect indicated by the velocity-based parameters dP/dtmax and VCF, and by the increase in V0 (p=0.039) and rightward shift of the PES–VES relation. This dissociation of force-based and velocity-based indexes is consistent with our prior study [16], and can be correlated with ryanodine induced alterations in the Ca2+ transient. Thus, ryanodine exerted significant negative inotropic and lusitropic effects without changes in HR or load.


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  		Table 1 Effect of ryanodine on baseline hemodynamic parameters

 
3.2 Effect of ryanodine on mechanical restitution
Fig. 1 displays analog tracings of a partial restitution sequence from a representative animal prior to and after the administration of ryanodine. Note the reduction in maximal dP/dt after ryanodine. In this animal, after ryanodine administration there was fusion of the extrasystole with the preceding control beat at the ESIs shown. Fig. 2 shows the technique of beat subtraction to derive the true extrasystolic response from a fused extrasystolic beat as described above. This phenomenon was seen at small ESIs in all but one of the animals after ryanodine administration and was secondary to ryanodine-induced prolongation of beat duration [14]. Fig. 3A and B show mechanical restitution curves from two different animals before and after ryanodine. Both panels relate normalized SBE to the ESI. In the animal in Fig. 3A, ryanodine accelerated MR with the time constant (TC) decreasing from 85.4 to 63.7 ms. Due to faster restitution kinetics after ryanodine, achievement of maximal contractile response occurs earlier, with a reduction of maximal contractile response (CRmax) from 126.5 to 111.9%. In some animals (four of the twelve dogs), MR became biphasic after ryanodine with a gradual late reduction in contractile response at long ESIs, as shown in Fig. 3B. This animal also displayed acceleration of early MR kinetics (TC decreasing from 32.6 to 22.5 ms) after ryanodine but with a late phase monoexponential decline (TC 307.9 ms). Group data for time constants of mechanical restitution (TCM), CRmax, and ESI0 under control conditions and after ryanodine are listed in Table 2. Four dogs displayed a biphasic MR curve after ryanodine as shown in Fig. 3B. Ryanodine produced significant acceleration of early MR kinetics (TCM1, p=0.032) and reduced CRmax (p=0.022), regardless of the presence or absence of a late phase. ESI0 was unchanged after ryanodine (p=0.87).


Figure 1
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  		Fig. 1 Analog tracings from a representative animal showing beats at two extrasystolic intervals (ESIs of either 240 or 300 ms) prior to and after the administration of ryanodine. The extrasystoles were delivered following several beats at the basic cycle length of 375 ms. After ryanodine, there is fusion of the extrasystolic beat with the preceding control beat due to beat prolongation (see text). LV, left ventricular; dP/dt, first derivative of LV pressure; ECG, electrocardiogram; AP, anterior–posterior; SL, septal–lateral; LA, long axis.

 

Figure 2
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  		Fig. 2 Illustration of the technique of beat subtraction. Beat duration is prolonged after ryanodine with a slowing of the kinetics of both contraction and relaxation (see text). This left ventricular (LV) pressure trace from one animal after ryanodine administration shows fusion of the extrasystolic beat with the immediately preceding control beat. To derive the true mechanical contribution of the extrasystole, the extrasystolic beat starting pressure was subtracted from the fused beat to yield a subtracted representation of the actual extrasystolic response.

 

Figure 3
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  		Fig. 3 Mechanical restitution (MR) curves from two different animals before and after ryanodine. Normalized single beat elastance (SBE) is plotted as a function of the extrasystolic interval (ESI). (A) After ryanodine there is acceleration of MR kinetics, earlier achievement of maximal response, and reduced maximal contractile response (CRmax). (B) In this animal, ryanodine resulted not only in acceleration of early MR kinetics, but also a slow late phase monoexponential decline in contractile response; TC, time constant.

 

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  		Table 2 Effect of ryanodine on mechanical restitution parameters

 
3.3 Effect of ryanodine on relaxation restitution
Fig. 4 shows RR curves from one animal before (A) and after ryanodine (B). Normalized relaxation responses (inverse of dP/dtmin) are plotted against the ESI. Ryanodine prolonged the kinetics of early RR (TCR1 41.1 versus 26.3 ms) and, in this animal, prolonged late phase kinetics as well (TCR2 161.2 versus 91.9 ms). Table 3 lists group data for RR time constants and plateau responses (RR1max and RR2max) for the early and late phases of RR before and after ryanodine. Ryanodine significantly slowed early RR (TCR1, p=0.034) without changing late RR kinetics (TCR2, p=0.47). The asymptotic plateau of the early phase was unchanged by ryanodine (RR1max, p=0.33), while that of the late phase was significantly decreased (RR2max, p=0.0019). Thus, ryanodine produced divergent effects on the kinetics of early MR and RR.


Figure 4
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  		Fig. 4 Relaxation restitution (RR) curves from one animal before (A) and after (B) ryanodine. Relaxation response is assessed using the normalized inverse of dP/dtMIN and plotted as a function of the extrasystolic interval (ESI). Under normal conditions, RR is biphasic with an early phase rapid recovery (R1) and a late phase of relaxation slowing (R2). In this example, there is slowing of both early and late phase kinetics after ryanodine, with the early phase time constant (TCR1) increasing by nearly 60% and late phase time constant (TCR2) by 75%.

 

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  		Table 3 Effect of ryanodine on relaxation restitution parameters

 
3.4 Effect of ryanodine on the force-frequency response
Fig. 5 shows EES values at heart rates of 120, 140, 160 and 180 bpm from a representative animal before and after ryanodine. The increase in EES with increasing heart rate is exaggerated after ryanodine. Table 4 shows group data for EES at the four heart rates. A pronounced FFR was present both at control and after ryanodine as indicated by the highly significant increases in EES at 140, 160 and 180 bpm as compared to 120 bpm (p<0.0167). EES was significantly greater at the highest heart rates of 160 and 180 bpm after ryanodine as compared to control (HR 160 bpm, p=0.020; HR 180 bpm, p=0.034), indicating amplification of the FFR despite an overall negative inotropic effect at baseline (Table 1).


Figure 5
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  		Fig. 5 Effect of ryanodine on the force-frequency response (FFR) in a representative animal. End-systolic elastance (EES) values are shown at heart rates of 120, 140, 160 and 180 bpm before and after ryanodine. Ryanodine markedly amplified the FFR despite a lower EES at baseline.

 

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  		Table 4 Effect of ryanodine on the force-frequency response

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
This study provides a comprehensive evaluation of the effects of selective impairment of physiologic SR Ca2+ release induced by ryanodine on force–interval and, for the first time, relaxation–interval relations in the intact animal. The findings demonstrate that ryanodine: (1) accelerates MR, reduces maximal contractile response, and, in a minority of subjects, uncovers a second late phase of decreasing contractile response; (2) amplifies the FFR despite reducing baseline inotropy and (3) consistently slows the kinetics of early RR, but not late RR, and reduces the late phase plateau of relaxation response. These findings suggest that in the intact animal, SR Ca2+ handling is a primary determinant of both MR and early RR, but not late RR. Conversely, augmentation of the FFR underscores the central role of SL Ca2+ influx in producing frequency potentiation.

4.1 Ryanodine effects on mechanical restitution
As described by Bers [1], the recovery of mechanical response during early MR is likely related to several factors including: (1) movement of Ca2+ from the longitudinal to junctional SR, an extremely rapid process via simple diffusion, (2) recovery of SL Ca2+ channels and inward Ca2+ current (ICa) from inactivation, a relatively fast process with a time constant of approximately 50–100 ms in isolated muscle [17, 18], and (3) recovery of the SR Ca2+ release channel from inactivation, a slower process with a time constant of 700 ms in skinned Purkinje fibers [19]. Under normal conditions, SR Ca2+ release channel recovery is probably the rate limiting mechanism governing MR with the more rapid process of SL Ca2+ channel recovery playing a lesser role [1, 3, 4].

Ryanodine binds selectively to the SR Ca2+ release channel in a dose-dependent fashion [20], without direct effects on the SR Ca2+ uptake pump [21]or myofilament Ca2+ sensitivity [22]. The dose administered in this study corresponds to nanomolar serum levels [14], a concentration which results in SR Ca2+ leak and depletion, reducing the capacity of the SR to accumulate Ca2+ during diastole [23]. As seen in Fig. 3 and Table 2, ryanodine resulted in acceleration of early MR and reductions in CRmax in the intact animal. These findings are in accordance with prior studies in isolated ventricular muscle [3, 4, 24]demonstrating that impairment of normal SR Ca2+ release with ryanodine in either micromolar [2, 3]or nanomolar [24]concentrations abbreviates MR. Wier and Yue [3]have postulated that this is due to the increased importance of SL Ca2+ channel recovery in determining this process in the presence of a dysfunctional SR, and that the time course of early MR is shortened due to faster recovery kinetics of SL Ca2+ current as compared to SR Ca2+ release [1, 3, 17, 18]. Also, as the SR no longer effectively accumulates Ca2+, smaller quantities of activator Ca2+ derived from the SL can trigger contraction [3–5].

In some animals, there was a second late phase of MR with a gradual decrease in contractile response at long cycle lengths (Fig. 3B and Table 2). This finding is also consistent with isolated papillary muscle studies demonstrating accentuation of the late phase decline in contractile response at long ESIs (>1000 ms) in the presence of either micromolar [4]or nanomolar [23]concentrations of ryanodine. This phenomenon is probably the result of impaired SR Ca2+ storage and more pronounced SL Ca2+ efflux at longer ESIs [4, 23]. Conversely, this phase is attenuated by ouabain [4], an inhibitor of the SL Na+/K+ pump and Ca2+ efflux via Na+/Ca2+ exchange, again indicating its dependence on SL Ca2+ efflux. Since very long cycle lengths cannot be easily assessed in the intact animal due to the presence of sinus escape, only one-third of the animals in this study demonstrated this late phase. Thus, analogous to isolated papillary muscle, the results of this study are consistent with ryanodine induced transformation of myocardial Ca2+ handling from being predominantly SR dependent to predominantly SL dependent.

4.2 Ryanodine effects on the force-frequency response
In isolated cardiac muscle and myocyte preparations with positive FFRs, increased stimulation frequency is associated with increased magnitude and slower inactivation of ICa [25], increased peak systolic and end-diastolic Ca2+ [26, 27], increased intracellular Na+ activity favoring Ca2+ influx via SL Na+/Ca2+ exchange [2, 28], and resultant increased SR Ca2+ loading and release [2, 29]. Thus, the FFR is associated with increased transsarcolemmal Ca2+ flux, increased intracellular Ca2+ concentration, and increased SR Ca2+ content available for myofilament activation. In the intact animal, the FFR is an important modulator of cardiac function during exercise [30]and acts synergistically with catecholamines [31], although its expression during baseline conditions is variable depending on the experimental preparation and parameters used to measure contractility [8, 10, 30–32]. As seen in Fig. 5 and Table 4, the animals in this study displayed significant FFRs as assessed using EES, in the presence of anesthesia and autonomic blockade.

After ryanodine, the FFR was amplified with significantly greater contractile responses at high heart rates as compared to control (Table 4). Similar results have been reported by other investigators in conscious dogs without autonomic blockade [32]. As described above, the production of SR Ca2+ leak by ryanodine reduces the ability of the SR to buffer changes in intracellular Ca2+ [3], and, as a result, smaller amounts of Ca2+ entering the cell via the SL produce larger changes in intracellular Ca2+ which are capable of triggering contraction [5]. If the FFR is ultimately a reflection of increased intracellular activator Ca2+ originating from transsarcolemmal flux, amplification of the FFR after ryanodine may be due to an exaggeration of the accompanying physiologic increase in intracellular Ca2+ concentration due to the presence of a dysfunctional SR. Indeed, studies in isolated papillary muscle [28]have shown that the frequency potentiation is greatest at lower levels of SR Ca2+ loading such as can occur after high nanomolar [5]or low nanomolar [28]ryanodine concentrations. This may be related both to increased end-diastolic Ca2+ levels [26]and a shift to the steeper region of the pCa–tension curve with higher stimulation rates, as well as increased availability of SR Ca2+ release due to less Ca2+ leakage with shorter intervals between beats. Of note, in species with a negative FFR, ryanodine in either nanomolar [6]or micromolar [26]concentrations converts the FFR to a positive one, indicating that unmasking the SL contribution to the FFR isolates a positive FFR component in all species. This concealed positive FFR is not associated with increased SR Ca2+ release upon higher rates of stimulation [26], but is correlated with increased intracellular Ca2+ levels. Thus, akin to MR, FFR amplification is consistent with increased importance of SL Ca2+ handling mechanisms after ryanodine.

4.3 Ryanodine effects on relaxation restitution
As seen in Fig. 4 and Table 3, ryanodine slowed the kinetics of early RR without changing the late phase time constant. By analogy with ryanodine effects on MR and the FFR, this suggests that early RR is dependent on SR Ca2+ handling, and that after ryanodine induced reduction of SR Ca2+ accumulation capacity due to SR Ca2+ leak, secondary SL Ca2+ uptake systems with slower kinetics assume a more prominent role in relaxation. Both major SL Ca2+ efflux systems, the SL Ca2+ pump and Na+–Ca2+ exchange have much slower operating kinetics than SR Ca2+ uptake pump [33]and would be consistent with this phenomenon. Late phase kinetics were unchanged by ryanodine, implying that this phase is dependent on factors external to the SR. This could involve SL efflux mechanisms whose capacity for Ca2+ efflux is limited and exceeded at longer cycle lengths resulting in late slowing of relaxation rates, the kinetics of which are unchanged after selective impairment of SR function. Interestingly, the second phase plateau was reduced after ryanodine indicating that relaxation rates are closer to capacity during stimulation at the basic cycle length. This effect may be the result of attenuation of load induced slowing of relaxation at long cycle lengths by ryanodine as demonstrated by Lew [34].

This study must be examined in light of methodological limitations. As in previous studies [7, 11], the V0 determined at the basic cycle length was used to calculate SBE for extrasystolic beats. Although steady-state increases in HR change V0, the effect of changes in VLV on end-systolic pressure occur over several beats [35], and would be minimized given the transient alteration of pacing interval used to produce test pulses. Additionally, as in many prior studies, our analysis assumes linearity of the PES–VES relation. In the intact canine model, Little et al. [36]have shown that although a slight, but consistent curvilinearity of the PES–VES relation exists regardless of inotropic state, this degree of nonlinearity does not prevent the relation from being well approximated by a straight line. In this study, the linear regression correlation coefficients were high, and it is doubtful that a significant quantitative error affected the analysis.

In summary, I have shown that ryanodine infusion results in acceleration of MR, reduction of maximal contractile response, slowing of early RR without changes in late RR kinetics, and amplification of the FFR. This modulation of force–interval and relaxation–interval behavior is consistent with the loss of SR dependence of intracellular Ca2+ handling and increased importance of SL Ca2+ transport mechanisms. MR, RR and the FFR and their responses to ryanodine can serve as powerful mechanistic tools to comprehensively define intracellular Ca2+ handling in vivo, facilitating the identification of underlying abnormalities in Ca2+ dynamics in the intact animal.

Time for primary review 23 days.


   Acknowledgements
 
This work was supported by a Grant-in-Aid from the American Heart Association, and an institutional grant from the South Texas Health Research Center of the University of Texas Health Science Center. The author gratefully acknowledges the excellent technical assistance of Danny Escobedo and Cindy Ramirez. The author thanks Dr. Gregory Freeman for his helpful insights regarding the manuscript. Dr. Prabhu is an Established Investigator of the American Heart Association.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Bers DM. Excitation–contraction coupling and cardiac contractile force. Dordrecht: Kluwer, 1991:155–158.
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