ELECTROMECHANICAL EFFECTS OF CYCLOPIAZONIC ACID ON MYOPATHIC HUMAN AND HAMSTER VENTRICULAR MYOCARDIUM

Jeng Wei¹, Hsiang-Cheng Liu³, Fan-Yen Lee², Cheng-I Lin^3
1Cheng-Hsin Rehabilitation Medical Center, Taipei, ²Chang-Gung Hospital Kaohsiung Medical Center and  ³Departments of Pharmacology & Biomedical Engineering, National Defense Medical Center,
Neihu 114,Taipei, Taiwan

Abstract: Our aim was to study the role of the sarcoplasmic reticulum (SR) in the abnormal electromechanical activity of ventricular tissue from myopathic Syrian hamster (Bio 14.6, 17~27 and 39~43 weeks) and explanted human failing hearts (n=8). Action potentials (APs) were recorded by means of a microelectrode technique, and force by a force transducer. Post-rest potentiation of contraction (PRPC), a measure of the SR-Ca²⁺ pumping activity, was determined after different rest intervals (2~60 s), in the absence and presence of 10 μM cyclopiazonic acid (CPA, a specific inhibitor of the SR Ca²⁺-ATPase). In human myopathic myocardium, the curve of PRPC-rest interval peaked at longer intervals as compared to that of the hamsters. CPA markedly depressed the relation throughout the curve and induced contracture at high driving frequency but did not generate spontaneous AP during rest interval. The present results show that in dilated myopathic human ventricular myocardium, mechanical behavior show similarity and differences with the myopathic Syrian hamster model. There are peculiarities that human myopathic ventricular tissue does not share with the hamster model.

INTRODUCTION

Dilated cardiomyopathy is characterized by an abnormal electromechanical activity. Several animal models have been used to explore the cellular mechanisms underlying the abnormal function of myopathic hearts. An animal model, the Syrian hamster (Biobreeders Bio 14.6) with inherited cardiomyopathy [1], has been shown to have defective intracellular Ca²⁺ handling [2], which may predispose the myopathic heart to Ca²⁺ overload and to triggered tachyarrhythmias [3,4].

The general aim of the present experiments was to analyze the role of possible abnormalities of Ca²⁺ uptake by the SR in the electromechanical performance of ventricular tissue of myopathic hamster as well as of human myopathic hearts. In specific, the modifications of the action potential and twitch after a fixed period of quiescence (post-rest potentiation of contraction, PRPC): an impaired Ca²⁺ uptake by the SR would be reflected in a smaller post-rest contraction [5]. In addition, the effects of the inhibition of SR Ca²⁺ uptake by cyclopiazonic acid (CPA) [6] on the mechanical performance of healthy and myopathic ventricular muscle to determine whether the inhibition is enhanced by the disease.

The interest of our study of the myopathic hamster resides in the fact that the information sought may provide useful insights in the mechanisms of altered function present in the related human pathological condition, namely, idiopathic dilated cardiomyopathy [7]. And the interest of the study of ventricular tissue from human myopathic heart affected by idiopathic dilated myopathy resides in the fact that it can be determined whether and to what extent the results obtained in our animal model apply to human tissue.

METHODS

Hamster ventricular tissue preparations: Three groups of Syrian hamsters were used: (a) 18 age-matched healthy hamsters (F1B) were obtained from Biobreeders (Fitchburg, Mass., USA) and served as control; (b) 18 male myopathic hamster (Bio 14.6) at age of 16 weeks were obtained from the same source. Eight of myopathic hamsters were used at the age of 17~27 weeks; and the remaining 10 myopathic hamsters were used at age of 39~43 weeks.

The animal was anesthetized with sodium pentobartibal (50 mg/kg, intraperitoneally) and heart was quickly removed through thoracotomy. The maximum ventricle circumference was measured in the older hamsters. The average value (mean±SEM) for myopathic hamsters (21.2±0.4 mm/100 g body weight) was significantly larger (+32%) than those for F1B (16.0±1.1 mm/100 g). Papillary muscles (diameter around 1.5 mm) were obtained from the left ventricle and were perfused with Tyrode solution of the following composition (in mM): NaCl 137, KCl 4, MgCl₂ 0.5, NaHCO₃ 15, NaH₂PO₄ 0.5, CaCl₂ 2.7 and dextrose 5.5. The solution was gassed with a mixture of 97% CO₂ and 3% CO₂, yielding a pH around 7.4 at 37 ^oC.

Human ventricular tissue preparations: Human ventricular trabecular muscles (about 1.5 mm in diameter and 5-8 mm in length) were obtained from the right ventricle of 8 patients with idiopathic dilated cardiomyopathy (7 male and 1 female, age 37±7 years) who underwent heart transplantation at Cheng-Hsin Medical Center. Informed consent was obtained from each patient. The human tissue was studied under the same experimental conditions as the hamster tissue.

Membrane potentials and contraction recording: One end of the preparation was pinned to the bottom of the tissue bath and the other end was tied to a force displacement transducer (Grass FT03C) with a resting tension of about 150 mg. The action potentials (APs) and contractile force were recorded as described previously [4]. Cyclopiazonic acid (CPA) and all other chemicals were obtained from Sigma Chemicals (St. Louis, Mo., USA).

Post-rest potentiation of contraction: Post‑rest potentiation of contraction  In cardiac muscle, the force of contraction increases after a transient interruption of drive [5]. The increase in contraction amplitude after rest intervals longer than the basic stimulation interval is referred to as PRPC. 

Statistical analysis: Statistical analysis Values are expressed as means ± SEM. One-way ANOVA and Student t test were used for statistical analysis. A P value less than 0.05 was regarded as statistically significant.

RESULTS

In Figure 1, it is shown that while PRPC subsided gradually in ventricular myocardium of myopathic hamster (as it did in healthy hamster), in human myopathic myocardium PRPC lasted only one beat. The relationship between PRPC and rest interval was similar in younger healthy and myopathic hamster, but the curve of the older myopathic muscle was obviously shifted downward. CPA (10 μM) decreased predominantly the ascending part of the curve in both the healthy and the myopathic hamster myocardium and could induce spontaneous AP during drug exposure or after washout.

In human myopathic myocardium, the curve of PRPC-rest interval peaked at longer intervals (40-60 s) as compared to that of the hamsters (10-20 s). CPA markedly depressed the relation throughout the curve and increased the diastolic force (from 25± 8 to 95±28 mg, P<0.05) at high driving frequency (180/min) but did not induce spontaneous AP during rest interval.

DISCUSSION

Our results suggest the following major findings: (a) the PRPC seems impaired in both older hamsters and human tissues from ventricles affected by dilated cardiomyopathy; (b) the impairment is increased by the inhibition of calcium uptake into the SR by CPA; (c) the impairment is exaggered by CPA; (d) PRPC is a function of the duration of the pause and becomes smaller as a function of the progress of disease; (e) there are several differences in the electrical and mechanical behavior of the myopathic tissues of the hamster and the humans, both in the steady state and after a pause.

We conclude that an impaired function of the SR contributes to the progressive deterioration of ventricular function in dilated cardiomyopathy and that electro-mechanical behavior of ventricular myocardium of patients affected by dilated cardiomyopathy shows similarity and differences with the myopathic Syrian hamster. The similarities include the dependence of PRPC on the driving rate and the depression of PRPC by the CPA inhibition of Ca²⁺ uptake into the SR. The differences in human tissue include a greater development of resting tension at faster rates (especially in the presence of CPA), the different shape of the relation between PRPC and rest interval at longer rest intervals and the more marked depression of this relation by CPA.

A more general conclusion is that, while animal models provide interesting and very valuable insights for the understanding of human disease, only the results in the human specimens provide direct evidence for the abnormalities of function caused by disease.

 

Figure 1. Post-rest potentiation of contractile force (PRPC) in ventricular muscles of a myopathic hamster (Bio 14.6, 43 week-old) (panel A) and an explanted human heart (panel B). The action potential and contractile force were recorded at 120/min in A and 60/min in B, before and after a 20 s rest interval. Electrical stimulation was interrupted between the downward arrow and upward arrows. Note different time scales for panels A and B.

Acknowledgements: The present work was supported by grant NSC-90-2314-B-350-001 from the N.S.C. and grant  90-02 from C.H.R.M.C., Taipei, Taiwan, ROC.

REFERENCES

[1]  Homburger F. Myopathy of hamster dystrophy: history and morphologic aspects, Ann N Y Acad Sci, vol. 317, pp. 2-17, 1979.

[2]  Kuo TH, Tsang W, Wiener J. Defective Ca²⁺-pumping ATPase of heart sarcolemma from cardiomyopathic hasmster, Biochim Biophys Acta, vol. 900, pp. 10-16, 1987.

[3]  Waldo AL, Wit AL. Mechanisms of cardiac arrhythmias, Lancet, vol. 341, pp. 1189-1193, 1993.

[4] Lin CI, Yiu MY, Hwang HR, et al., Reentrant tachyarrhythmias in right atria of cardiomyopathic versus healthy Syrian hamster, J Biomed Sci, vol. 6, pp. 399-408, 1999.

[5]  Bers DM, Bassani RA, Bassani JW, et al., Paradoxical twitch potentiation after rest in cardiac muscle: increased fractional release of SR calcium, J Mol Cell Cardiol, vol. 25, pp. 1047-1057, 1993.

[6]  Chiesi M, Wrzosek A, Grueninger S. The role of the sarcoplasmic reticulum in various types of cardiomyocytes, Mol Cell Biochem, vol. 130, pp. 159-171, 1994.

[7]  Ishino K, Botker HE, Clausen T, et al., Myocardial adenine nucleotides, glycogen, and Na,K-ATPase in patients with idiopathic dilated cardiomyopathy requiring mechanical circulatory support, Am J Cardiol vol. 83, pp. 396-399, 1999.