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Cardiovascular Research 2004 61(1):77-86; doi:10.1016/j.cardiores.2003.09.022
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Quantification of the rat left ventricle force and Ca2+–frequency relationships: similarities to dog and human

David G Taylora, Leonard D Parilakb, Martin M LeWinterc and Harm J Knot*,a,b,c

aDepartment of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610, USA
bDivision of Cardiovascular Medicine, University of Florida, Gainesville, FL 32610-0267, USA
cCardiology Unit, Department of Medicine, University of Vermont, Burlington, VT 05401, USA

* Corresponding author. Department of Pharmacology and Therapeutics, PO Box 100267, Gainesville, FL 32610-0267, USA. Tel.: +1-352-392-5317; fax: +1-352-392-9696. hknot{at}college.med.ufl.edu

Received 17 July 2003; revised 5 September 2003; accepted 15 September 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Objective: To measure and quantify the force–frequency (FFR) and Ca2+–frequency (CaFR) relationships in isolated rat left ventricular (LV) muscle at physiological heart rates and compare the obtained FFR to that measured in larger mammalian muscle from dog and human using the same experimental protocol. Methods: Rat papillary muscle was isolated from the LV of adult male Sprague–Dawley rats, and dog and human muscles were from free-wall LV biopsies, loaded with the Ca2+ indicator Fura-2, allowed to recover from isolation trauma and then subjected to direct electrical stimulation while measuring force production and intracellular Ca2+ transients. Results: We obtained a positive FFR between 1 and 4 Hz that is qualitatively similar to that found in isolated LV epicardial muscle strips from dogs and humans with normal LV function. The FFR reflects the cytosolic Ca2+ transients in amplitude. Isoproterenol yielded an enhancement in force, but flattening of the FFR, whereas cyclopiazonic acid caused depression of FFR amplitude without changing frequency-dependent shape. Conclusion: We describe an experimental protocol that consistently yields positive FFRs in rat, dog and human LV muscle at stimulation rates between 1 and 4 Hz, without significant qualitative differences. We attribute previously observed negative FFR in rat muscle to an increase in SERCA activity early after excision and preparation of the muscle strips.

KEYWORDS e-c coupling; Contractile function; Calcium (cellular); SR (function); Ventricular function


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
In most mammals, cardiac contractile force increases with an increase in stimulation frequency, a phenomenon known as the treppe effect or positive force–frequency relationship (FFR) [1]. The molecular basis of the treppe effect has been attributed to a net gain of circulation of Ca2+ in the cell and increased calcium-induced calcium release, by either increased Ca2+ entry through the L-type Ca2+ channels and/or a reduction in Na+/Ca2+ exchanger activity during diastole due to more numerous depolarizations per unit time [2], both coupled to greater capturing and Ca2+ loading of the sarcoplasmic reticular Ca2+ stores by enhanced SERCA activity [3].

In species whose contractile force more heavily depends on SR Ca2+ cycling for providing contractile Ca2+, such as the mouse and rat [4], the FFR regularly observed in experimental preparations has been negative in the range of 1–4 Hz [5,6]. This is in contrast to the positive FFR observed in larger mammals such as dog and human in that frequency range, despite the remarkable similarity in structure and primary sequence of all key proteins involved in Ca2+ cycling and force production among mammals.

Although the mouse and rat are the most common laboratory models, this fact has slowed acceptance of these animals as a model for research into cardiac diseases in humans, for which a depressed FFR is often both symptomatic and diagnostic [3,7]. To explain the observations in rat, observers have postulated several parameters that can affect the in vitro FFR, including specimen size [8] and solution composition. Nonetheless, a positive FFR could be induced in the rat by altering experimental conditions that impair SR Ca2+ release, such as reducing extracellular Ca2+ [9] or ryanodine treatment [10]. Together, these results suggested that rat cardiac tissue is intrinsically capable of presenting the characteristic mammalian FFR if an appropriate experimental protocol were applied. Recently, using newer protocols and approaches, positive FFRs are being observed in the rat [11,12] and mouse [13,14] in cardiac muscle preparations. The shape of the FFR curve differs somewhat but not greatly from that seen in larger mammals in slope and optimal frequency (frequency of greatest systolic force) [3,15].

Although the mouse has become a popular subject for genetic manipulation, its use is limited for obtaining quantitative physiological measurements in vivo and in vitro. In contrast, several rat strains, such as the Spontaneously Hypertensive Rat (SHR) and Spontaneously Hypertensive/Heart Failure-Prone Rat (SHHF), which are currently available, are increasingly revisited [16] in cardiovascular research. In fact, these rat models have been the basis in the past for the development of many cardiovascular drugs that are on the market today. For these and other reasons, several new transgenic rats with disease profiles aimed at mirroring human pathology have been generated [17]. Although most pathology lies in the left ventricle, most studies in rats focused on right ventricle tissue for perceived technical reasons and limitations associated with left ventricular (LV) such as muscle strip dimension [8,11,13,14].

The goal of this study was to use a widely used protocol developed for human LV biopsy strips [3] and apply this to the study of isolated rat LV muscle to investigate whether this would reveal a positive FFR that is similar in shape to that seen in humans and dogs. We hypothesized that based on the sequence and functional similarity of proteins involved in Ca2+ cycling, the FFR in mammals should be comparable if they were obtained under the same experimental conditions. To this protocol, we added loading of the cytoplasm with the Ca2+ dye Fura-2, in the time set aside for equilibration, which allows simultaneous quantitative measurements of systolic and diastolic Ca2+ transients with force. We further used this protocol to study the effects of cardioactive drugs with known defined action on Ca2+ cycling to further elucidate the nature of the mechanisms underlying the ascending limb of the FFR and particularly the likely cause of the persistent negative FFR found in rat when the experiments are performed within an hour of dissection of the preparation. We show that the rat FFR is remarkably similar to that in humans and dogs when the same experimental protocol is used. Our data further suggest that the negative FFR in rat, when taken early after dissection, is due to high post-isolation/dissection Ca2+ cycling caused by high activity of the SR SERCA Ca2+ pump.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1 Preparation and protocols
Twenty-seven male Sprague–Dawley rats (275–325 g; Harlan, age 9–11 weeks) were anesthetized with isoflurane; the hearts were excised, placed and further dissected in Krebs–Henseleit (KH) solution at 4 °C containing (in mmol/l): 127 NaCl, 2.3 KCl, 25 NaHCO3, 1.3 KH2PO4, 2.5 CaCl2, 0.6 MgSO4, 11 Glucose, 30 2,3-butanedione monoxime (BDM) (Sigma, St. Louis, MO, and Fisher Scientific, Fair Lawn, NJ), with 10 units/l insulin, saturated with 95% O2–5% CO2, pH=7.4. LV papillary muscles were removed and placed in the horizontal quartz flowchamber of a Muscle Research System (Scientific Instruments, Heidelberg, Germany). Both ends of the muscle were held by stainless-steel tweezers, one attached to a Grass SDJ9 stimulator (Grass Instruments, West Arwick, RI), and the other to a force transducer. The cross-sectional area between the tweezers was measured via a micrometer. Muscles wider than 1.6 mm were excluded.

Human subjects were recruited from patients with coronary artery disease referred for CABG. Inclusion criteria were: no prior MI, and normal LV function and mass as assessed by echocardiography. Patients included four females and six males aged 66±7 years. At CABG, sub-epicardial biopsies were obtained about 5 min after cardiac arrest [18]. The surgeon excised a strip of myocardium measuring ~12 x 2 x 2 mm from the anterior LV wall. The exact site depended on the distribution of epicardial vessels and fat. The specimen was immediately immersed in a pre-oxygenated KH solution containing 30 mmol/l BDM and soaked for at least half an hour before subsequent dissection and performance of in vitro studies [18].

Mongrel dogs of different age and weight were purchased for studies on LV function, unrelated to this study, from USDA registered vendors. Cardiac tissue was harvested as described for humans from dogs that did not receive cardiodepressant drugs, with approval from the local IACUC.

In all preparations, force measurement was performed isometrically in oxygenated KH without BDM at pH=7.4, 30 °C. Flow rate was ~1.5 ml/min. Stimulation pulses were bipolar and 5 ms in duration. After 5 min in KH, stimulation voltage was set to ~30% above threshold. After 30 min of equilibration at 0.15 Hz, the muscle was gently stretched to the length of maximum systolic force (L0). An initial FFR curve was performed at 0.2–6 Hz. The muscle was stimulated at each frequency for 1 min prior to recording.

The preparation was loaded with Fura-2 by recirculating oxygenated KH solution containing 5 µmol/l Fura-2 AM ester (Molecular Probes, Eugene, OR) with 0.5% dimethyl sulfoxide and 0.2% cremophor (to increase Fura-2 solubility), and 0.01 N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, a metal chelator that has been shown to enhance cardiac recovery after ischemia and reperfusion [19,20]) at 30 °C for 3.5–4 h, under continuous stimulation at 0.15 Hz. After loading, the muscle was re-equilibrated for 30 min in KH solution at 0.15 Hz, then 0.2 Hz for 5 min before an FFR was performed as described above.

The emission of Fura-2 excited alternately at 340/380 nm light was collected by a photometer unit and sampled by a signal sorter at 2–4 ms per ratio. Developed force (in mN) was recorded in OXC software and raw force and Ca2+ ratio transients in IonWizard v4.44 (IonOptix, Milton, MA) at 500 Hz.

2.2 Drug studies
After the 30-min Fura-2 washout/equilibration, muscles were stimulated at 0.2 Hz for 5 min in KH, then in KH with the predetermined EC50 of isoproterenol or CPA until peak systolic force stabilized. The FFR was then performed as described above.

2.3 Post-rest potentiation (PRP)
After 2–5 min of equilibration at a given frequency, stimulation was stopped for given number of seconds, then restarted. The value of the first systole was compared to the mean systolic value of five contractions preceding the rest period.

2.4 Data analysis
Forces were normalized to mN/mm2 tissue. Diastolic tension at 0.5 Hz was set as 0 mN baseline diastolic force. Force transients were analyzed with Ca2+ transients in IonWizard 5. Student t tests or ANOVA were performed to assess statistical significance (at 95% confidence limit) in Prism 3.0, and graphs were made in Origin 6.0. Data are shown as mean±S.E.M.

2.5 Ethical use of animal and patient material
The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996) and was approved by the Institutional Animal Care and Use Committee of the University of Florida. The institutional review board approved the collection and use of human material, and all patients gave informed consent.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1 Systolic force frequency relationships in rat, dog and human
The initial FFR in rat obtained within 60 min after dissection, following equilibration is negative and characterized by very high forces as shown in Fig. 1A. Peak force declined from 11.85±0.81 mN/mm2 at 0.2 Hz to 5.21±0.32 at 6 Hz. In contrast, after the 3–4 h Fura-2 loading time, developed force had dropped at all frequencies, but particularly below the optimal frequency of 3–4 Hz. As a result, the FFR revealed a markedly positive ascending limb. The FFR had a steep increase in developed force from 0.5 Hz to 3 Hz (0.5 Hz: 2.47±0.24 mN/mm2, 1 Hz: 2.66±0.24, 2 Hz: 3.87±0.34, 3 Hz: 4.96±0.42, n = 9). Developed force peaked at 3–4 Hz (4 Hz: 4.96±0.47), implying that the optimal frequency lay between 3 and 4 Hz, and declined slightly beyond 4 Hz (5 Hz: 4.53±0.45, 6 Hz: 4.01±0.39) (Fig. 1A,B). Diastolic force is shown in Fig. 1A. The FFR for diastolic tension increased slightly by 0.13±0.04 mN/mm2 from 0.5 to 2 Hz, remained unchanged from 2 to 4 Hz (4 Hz: 0.13±0.03) and slightly increased from 4 to 6 Hz (5 Hz: 0.23±0.05, 6 Hz: 0.41±0.09) compared to systolic force.


Figure 1
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  		Fig. 1 Systolic force–frequency relationships in rat, dog and human. (A) FFR of isolated papillary muscle after 45 min of equilibration (prior to Fura-2 loading) and after 3.5–4 h in recirculating KH with Fura-2 AM (post-Fura-2 loading). (B) FFR in experimental versus control conditions after 3.5–4 h in Fura-free recirculating KH compared to Fura-containing KH. (C) FFR of isolated rat papillary and human (n = 10) and dog (n = 6) LV myocardial strips using same experimental protocol. (D) Plot of heart rate at maximal force versus percent potentiation of force over the force at 1 Hz.

 
To determine if this force decline was a consequence of the Fura-2 dye loading, which as a calcium dye has the potential to buffer cytosolic calcium, we performed experiments in which the KH physiological solution was recirculated without Fura-2 AM, but with TPEN and cremophor for 3.5–4 h. The resulting FFR was positive with an optimal frequency at 4 Hz and not different from the FFR in Fura-2 loaded tissue (1–6 Hz, n = 5, p<0.05, Fig. 1B).

We used the same protocol to measure the FFR in epicardial strips from dog and human left ventricle. In order to compare the FFR in rat to those obtained in dog and human, and because these strips originate from scalpel biopsies that are pared down, we converted all FFRs by normalization to the force at 1 Hz. The result is shown in Fig. 1C. In humans, the index of the heart-rate-induced increase in force between 60 and 180 bpm is the physiologically most significant and has been termed chronotropic contractile reserve (CCR) by Alpert et al. [3]. The CCR for rat, dog and human were similar, yet differed in amplitude and heart rate of maximum force (Table 1). The relation between optimum heart rate and CCR was linear (Fig. 1D).


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  		Table 1 Amplitude and shape of the FFR among species

 
3.2 Force and calcium frequency relationships in rat LV muscle
It is well accepted that force development closely mirrors the increase in cytosolic Ca2+ in cardiac muscle in health and disease. Fig. 2A shows a representative original experiment of simultaneous measurement of developed force and cytosolic calcium at stimulation frequencies from 0.5 to 6 Hz in isolated adult rat papillary muscle.


Figure 2
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  		Fig. 2 Force and calcium–frequency relationships in rat LV muscle. (A) Representative developed force (thick line) and cytosolic calcium (Fura-2 AM, thin line) tracings from 0.5 to 6 Hz over 335 ms from stimulation pulse. Systolic intracellular Ca2+ level increases with an increase in frequency from 0.5 to 3 Hz and is accompanied by an increase in developed force. Rate of decline in both force and calcium transients accelerates with an increase in frequency. Note that the Ca2+ ratio scale is exponential, where small changes at the top represent substantial changes in Ca2+. (B) Force– and calcium–frequency behavior shown as percentage of peak value at 0.5 Hz. (C) Peak developed force as a function of peak calcium in individual experiments.

 
Each force transient was preceded by a cytosolic calcium transient with a relative peak height corresponding to the relative peak height of the force transient. As frequency increased, force and calcium transient amplitude increased as shown in Fig. 2B while retaining their close relationship. The change in developed force was mirrored by a corresponding change in cytosolic calcium concentration. From 0.5 to 3 Hz, peak force doubled (196.6±22.9%) and calcium increased to 126.7±4.9% (n = 8). From 4 to 6 Hz, developed force fell to 157.1±21.0% and calcium fell to 120.9±6.7% the 0.5 Hz value. As first shown by Brixius et al. [21] and Gwathmey et al. [22], the relationship between peak force and peak Ca2+ is near linear and shown for all individual experiments in Fig. 2C.

3.3 Kinetics of force and Ca2+transients as a function of frequency in rat LV muscle
We performed further analysis of several components of the systolic force and calcium transients that are most likely to alter under pathological conditions. In all cases, calcium values are reflected in the subsequent force transient. Time from stimulus pulse to peak force declined significantly from 1 to 4 Hz and remained constant up to 6 Hz (0.5 Hz: 74±2 ms, 2 Hz: 67±2, 3 Hz: 62±2, 4 Hz: 60±1, 5 Hz: 60±1, 6 Hz: 59±1, p<0.0001), but was unchanged in the calcium transient (Fig. 3A).


Figure 3
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  		Fig. 3 Kinetics of force and Ca2+ transients as a function of frequency in rat LV muscle. (A) Time from stimulus to reach peak force and Ca2+. (B) Maximum velocities of force and Ca2+ in relaxation phase of the transients. (C) Time from peak to 50% relaxation from peak value. Force n = 9. Calcium n = 8. {dagger}p<0.05 from 0.5 Hz value. *p<0.05 from value of preceding frequency.

 
The relaxation velocity of force increased with frequency and closely mirrored the systolic FFR, whereas the Ca2+ return velocity, a direct indicator of SERCA activity, remained constant up to 2 Hz but then markedly increased between 2 and 6 Hz (Fig. 3B). The time of force and calcium transient to return to 50% of peak value (RT50) significantly accelerated with an increase in frequency (Fig. 3C). Speed of decline in calcium was significantly faster than the preceding frequency at all experimental frequencies 2 Hz and greater. Speed of decline in force was only significantly faster than the preceding frequency at 2 Hz and above (RT50 1 Hz: 124±4 ms, RT 2 Hz: 114±3, p<0.05), which was also the frequency of greatest incremental increase in peak force.

3.4 Effect of isoproterenol and cyclopiazonic acid on the FFR, Ca2+ and diastolic force
To further ensure that our results reflect normal mechanisms within the myocardium, we treated the muscle with drugs with defined cardioactive effects and proceeded to measure the FFR. These results are summarized in Fig. 4 (each drug n = 5). Representative developed force and calcium transient tracings of muscle treated with isoproterenol and CPA stimulated at 2 Hz are shown in Fig. 4A.


Figure 4
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  		Fig. 4 Effect of isoproterenol and cyclopiazonic acid on the FFR, Ca2+ and diastolic force. Force–frequency behavior of isolated papillary muscle in 100 nmol/l isoproterenol (n = 5) or 10 µmol/l cyclopiazonic acid (n = 5), compared with controls (n = 9); *p<0.05 from controls. (A) Representative systolic force and calcium tracings (Fura-2 AM) in papillary muscles in isoproterenol, cyclopiazonic acid and controls at 2 Hz. (B) Systolic FFR. (C) Diastolic FFR.

 
The β-adrenergic agonist isoproterenol, at a predetermined EC50 of 100 nmol/l, increased peak developed force at all frequencies, but especially at low stimulation rates, thereby flattening and eliminating the ascending limb of the FFR. Peak force was significantly higher, particularly at frequencies below the optimal frequency of ~3 Hz. Isoproterenol also shortened the calcium and force transients (Fig. 4).

The specific SERCA inhibitor cyclopiazonic acid, at a predetermined EC50 of 10 µmol/l, reduced both absolute and proportional increase in peak force compared to controls (Fig. 4B). Isoproterenol lowered (p<0.05, 1–4 Hz) and CPA raised (p<0.05, >3 Hz) diastolic force across most of the FFR (Fig. 4C).

3.5 Mechanistic investigations
To further investigate the possibility that high SERCA activity early post-dissection may represent one cause of the preload negative FFR, we performed two established measurements closely related to SERCA activity—force relaxation velocity (FRV) and PRP. Post-loading FRV increased 2.9-fold with frequency, yielding a relationship qualitatively similar to the post-loading FFR (0.5 Hz: 22.9±3 mN/s; 4 Hz: 66.2±8), whereas the pre-Fura-2-loaded FRV remained unchanged (0.5 Hz: 78.4±6.6; 4 Hz: 79.8±8.4) but significantly higher than post-loading at the lower frequencies. Isoproterenol in post-loading papillary muscles accelerated relaxation velocity at all frequencies (3.6-fold at 0.5 Hz: 82.5±9.3; 1.6-fold at 4 Hz: 110.1±11.6) (Fig. 5A).


Figure 5
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  		Fig. 5 Effect of time on SERCA activity. Indirect SERCA activity measurements in isolated papillary muscle after 45 min of equilibration (prior to Fura-2 loading) and after 3.5–4 h in recirculating KH with Fura-2 AM (post-Fura-2 loading) and in 100 nmol/l isoproterenol. (A) Relaxation velocity. Preload n = 7. Postload n = 9. Isoproterenol n = 5. *p<0.05 from post-load controls. {dagger}p<0.05 from pre-load. (B) Post-rest potentiation behavior at 5 s of rest. Preload n = 3. Postload n = 5. {dagger}p<0.05 from 2 Hz value.

 
Developed force was augmented by frequency in the post-Fura-2-loaded PRPs from 190% at 2 Hz to 320% at 5 Hz of the pre-rest steady-state force, whereas increasing frequency had no effect (from 150% at 2 Hz to 150% at 5 Hz) on potentiation prior to Fura-2 loading. These data strongly suggest that SERCA activity was already near maximal in pre-Fura-2-loaded muscle (Fig. 5B).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
In this paper, we present the first comprehensive qualitative and quantitative analysis of rat isolated LV muscle function, measured simultaneously as the FFR and Ca2+–frequency relationship (CaFR) for systolic and diastolic function, in small isolated papillary muscle strips in vitro using a protocol designed for larger mammals. Our results reveal a positive FFR and suggest that the reason for often observed negative FFR is at least partly related to enhanced Ca2+ cycling, driven by the SERCA pump, in tissue early post-dissection.

4.1 Comparison of the FFR in rat to that in dog and human
It is well accepted that the molecular bases for the shape and amplitude of the FFR are intimately related to the fate of intracellular Ca2. Therefore, it has been surprising to see differences between smaller and larger mammals in the FFR despite the extraordinary molecular similarity at the DNA and protein level of proteins involved in Ca2+ cycling. To illustrate this point, we performed a comparative analysis on the protein sequences available in GenBank as shown in Table 2. We hypothesized that based on the sequence and functional similarities, the FFR in mammals should be comparable if they were obtained under the same experimental conditions. We found that the FFR obtained in rat is strikingly similar to that seen in larger mammals with established positive force–frequency relationships, such as human and dog when done under the same experimental conditions (Fig. 1). The steep ascending limb of the FFR occurs over the same stimulation range (~0.5 to ~3.5 Hz) and shares a similar peak value (~200% of 1 Hz force). The rat FFR also has similar optimal frequency (~3 Hz) to the human FFR in this study and to other published human data [3,15,23–25] taken from nonfailing control myocardium in Tyrode's solution and are also similar to our canine data obtained in KH solution and similar to that from Bouchard and Bose [5].


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  		Table 2 Comparisons of amino acid sequences of major cardiac Ca2+-handling proteins in rat, human and dog

 
We examined stimulation frequencies within and beyond the normal physiological range of humans (~1–3 Hz) and rats (4–7.5 Hz [26]), where depression of the FFR in the 1–3 Hz (60–180 bpm) range is both diagnostic and symptomatic of disease in humans.

Taken together, these data suggest that similar mechanisms may underlie this part of the FFR in all three species. Normal cardiac physiology in humans, dogs and rats is very different, so quantitative differences in force output and optimum heart rate are expected at the level of EC coupling. Alpert et al. [3] have previously shown a remarkable linear correlation between optimum heart rate of the FFR and the amplitude of the FFR in humans with different degrees of cardiac disease using this protocol. We report here a similar finding for these parameters in human versus dog and rat using this protocol, further suggestive of a similarity in underlying mechanisms (Table 1).

4.2 Kinetics of force and Ca2+transients as a function of frequency in rat LV muscle
We used advanced transient analysis to further look into possible mechanisms underlying the positive FFR in rat heart. Kinetic analysis of the excitation phase of the force and Ca2+ transients revealed a marked frequency-dependent up-regulation of the amount of Ca2+ into the cytoplasmic space. As elegantly shown by Bers [4], in rat, the bulk of Ca2+ released for contraction is thought to originate from the SR. Previous studies in rat have demonstrated that the efficiency and gain of EC coupling is near optimal and that release from the SR is quantal and directly related to the activity of the L-type Ca2+ channel (DHPR) [27]. We suggest therefore that the increased amount of Ca2+ may result from an increase in the amount of "trigger" Ca2+ supplied by the DHPR. This remains to be proven and is the subject of current investigation.

Analysis of the relaxation phase of the force and Ca2+ transients reveal a marked increase in speed of lowering cytosolic Ca2+ above 2 Hz up to 6 Hz as well as speed of force relaxation as a function of stimulation frequency between 1 and 4 Hz. These observations most likely reflect the canonical belief that SERCA activity increases with frequency due to frequency-dependent activation of Calmodulin Kinase II [3,28].

4.3 Pharmacological effects on the rat LV FFR
Consistent with our hypothesis that the high forces at low frequencies are a result of enhanced Ca2+ cycling are our observations on the effect of isoproterenol. Isoproterenol mostly affected the low-frequency side of the FFR. The increase in force resulted in a flattened FFR with a "gull-shape" and enhanced force at all stimulation frequencies. The most likely mechanisms are the effect on the DHPR [29], leading to increased Ca2+ channel activity, in tandem with the stimulatory effects enhancing Ca2+ efflux from the SR, via the ryanodine sensitive Ca2+ release channels, and most notably increased SERCA activity, by phosphorylation of phospholamban, resulting in higher SR Ca2+ load.

Consistent with published data on the effect of CPA on the positive human FFR [30], CPA both lowered the peak and lengthened the decline of the calcium transient, indicating reduced SERCA pumping of cytosolic calcium into the SR resulting in depressed systolic force. CPA also depressed the systolic amplitude of the FFR and increased the diastolic FFR, consistent with previous studies in human heart muscle [30].

4.4 Mechanistic investigations/limitations and perspective
Notwithstanding our present study, one remaining question is why earlier studies consistently showed negative FFRs. Several extrinsic causes for the negative FFR have been suggested, including hypoxia or metabolic waste accumulation due to tissue size in in vitro observations, decreased SR Ca2+ load (due to Ca2+ efflux or irregular spontaneous RyR Ca2+ release) or release with frequency [11], and high [Na+]i slowing Ca2+ extrusion and causing the filling of the SR even at low frequencies [4].

Tissue size was found to have an impact on the slope of trabecula FFR by Gulch and Ebricht [8]. We observed inconsistencies in peak and diastolic force production in very wide muscles, but not below our limit of 1.6 mm. Adequate oxygenation of our preparation, even at higher rates, is inferred from the increase in SERCA activity (a major consumer of ATP) seen with frequency. Post-peak [Ca2+]i decline velocity (92% due to SERCA in the rat [4]) increased and RT50[Ca] quickened throughout the measured frequencies up to 6 Hz (Fig. 3B,C). Absolute force at 6 Hz also stayed well above the force obtained at the lowest frequency of 0.5 Hz.

While decreased SR Ca2+ release due to RyR or L-type Ca2+ channel inactivation between action potentials is a possible explanation for the descending FFR at 4 Hz and greater and is the subject of current investigation, it does not adequately explain the different observations regarding the rat FFR in previous studies. Both positive and negative rat FFRs have been observed in physiological solutions containing 135–152 mmol/l Na+ and 4.7–5.8 mmol/l K+ [5,9–11,31], suggesting that [Na+] cannot be the exclusive cause, although it may still contribute substantially to force amplitude due to its effect on Ca2+ load.

A likely candidate suggested by our present results is recovery from cold ischemia during excision of the heart and preparation of the muscle strips. Retrospectively, we examined the time from dissection to actual experiment in papers in which a positive FFR was seen in the rat. These times were found to be lengthy, exceeding 1–3 h [11,14]; and including Fura-2 loading, our experiments occurred almost 5 h post-dissection. With recovery time, the rat in vitro FFR experiences a reduction in developed force, mainly at lower frequencies, which "unmasks" the positive slope. Spurred by our isoproterenol data, we realized that near-maximal SERCA activity with little room for frequency-dependent increase would produce such a shape and investigated SERCA activity pre- and post-Fura-2 loading. As direct Ca2+ measurements are impossible prior to dye loading, we performed two established measurements to assess SERCA activity—relaxation velocities and post-rest potentiations (Fig. 5). Speed of force decline (which is largely due to removal of cytosolic Ca2+ by SERCA) was significantly faster pre-Fura-2 loading at frequencies up to 2 Hz. Post-rest systolic contractions have been shown to quantitatively measure SR Ca2+ loading and to augment with frequency in humans [32]. This feature is readily observed in rat LV post-Fura-2 loading but absent in the pre-loading FFR (Fig. 5).

The rat heart is metabolically very active in situ, with heart rates often exceeding 400 bpm [26], and more dependent upon SERCA [4] for force production. Whereas human and dog tissue were carefully excised from arrested and merely cooled (to ~15 °C) hearts on cardiopulmonary bypass and then further dissected at room temperature, rat hearts are typically quickly excised at regular heart rate and temperature and then immediately immersed in ice-cold KH buffer. The biochemical trauma of this brief warm/cold ischemia and the associated disruption of the Donnan equilibrium is unknown and the subject of current investigation. It is conceivable that more time is needed for the tissue to recover from this trauma caused by prolonged hypothermia.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
In conclusion, rat LV papillary muscle can augment force as a function of stimulation rate similar to larger mammals, such as dog and human when subjected to the same experimental protocol. The positive FFR is likely due to a corresponding increase in cytoplasmic Ca2+ and increased SR Ca2+ loading by increasing SERCA activity. In freshly isolated tissue, SERCA activity is very high and hence frequency-dependent increase is greatly diminished, but recovers with time. In conclusion, with the current protocol, the rat LV shares basic cardiac functional characteristics with dog and human that may render it a better laboratory model for cardiovascular diseases in which an altered FFR is a distinguishing feature than previously assumed.


   Acknowledgements
 
This study was supported by American Heart Association predoctoral fellowship 0315172B (D.G.T), AHA postdoctoral fellowship 0120362B (L.P.), AHA Scientist Development Grant 0030159N (H.J.K.) and NIH HL61556 & HL50287 (M.M.L.).


   Notes
 
Time for primary review 32 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

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