Cardiovascular Research

Cardiovascular Research 1997 34(1):91-98; doi:10.1016/S0008-6363(97)00034-5
© 1997 by European Society of Cardiology

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Copyright © 1997, European Society of Cardiology

Intracellular calcium levels are unchanged in the diabetic heart

Jen Z Yu^a, Brian Rodrigues^b and John H McNeill^b,*

^aCardiovascular Research Laboratory, Department of Pathology and Laboratory Medicine, Faculty of Medicine, The University of British Columbia, Vancouver, BC V6T 1Z3, Canada
^bDivision of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, BC V6T 1Z3, Canada

^* Corresponding author. Tel. +1 604 822-9373; Fax +1 604 822-8001.

Received 24 October 1996; accepted 9 January 1997

KEYWORDS Diabetes; Cardiomyopathy; Calcium, intracellular concentration; SR, calcium release

	1 Introduction

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Diabetes mellitus has been associated with cardiac disease that has been suggested to occur secondary to atherosclerosis of the coronary arteries, macroangiopathy and autonomic neuropathy [1]. However, a cardiac disease particular to diabetes has also been demonstrated to occur in the absence of the above factors. This ‘diabetic cardiomyopathy’ that includes cardiomegaly, left ventricular dysfunction and clinically overt congestive heart failure has been strongly suggested by epidemiological, clinical and experimental studies [2–7]. The etiology of diabetic cardiomyopathy is complex and a number of factors have been suggested to be involved in the development of this disease state. These include: (a) changes in cardiac metabolism, (b) abnormal vascular sensitivity and reactivity to various ligands, (c) increased stiffness of the ventricular wall associated with perivascular thickening of basement membrane and interstitial accumulation of glycoprotein and insoluble collagen, and (d) abnormalities of various proteins which control ion movements, specifically intracellular calcium [6].

Streptozotocin (STZ) induction of diabetes in rats is the most commonly used animal model of diabetes. The chemical structure of STZ comprises a glucose molecule with a highly reactive nitrosourea side-chain that is thought to initiate its cytotoxic action. The glucose moiety directs this agent to the pancreatic β-cell [8]. STZ selectively destroys pancreatic β-cells and produces a diabetic state, the severity of which can be varied by altering the dose of STZ. With time (4–6 weeks), rats so treated develop biochemical and functional myocardial abnormalities which are the result of the drug-induced metabolic changes rather than a direct effect of the drug itself [6]. Thus, rats made diabetic with STZ exhibit depressed left ventricular contractility, diminished ventricular compliance and decreased inotropic and chronotropic responses to certain drugs in isolated cardiac preparations [4, 9, 10]. Changes in contractile properties of the heart have also been found in vivo in both anaesthetised [11]and conscious [12, 13]STZ-diabetic rats. Considering that the diabetic rat does not develop atherosclerosis, the in vivo and in vitro abnormalities in contractility and metabolism reflect changes in the function of the myocardial cell [14]. Hence, similar to the human condition, the STZ diabetic rat is a useful model to elucidate the pathogenesis of diabetic cardiomyopathy. In this review, evidence is presented that basal calcium ([Ca²⁺]_i) levels in both quiescent and electrically-stimulated cardiac cells from STZ diabetic rats is unchanged and may not reflect the cardiac abnormalities observed in STZ diabetes.

	2 [Ca2+]i mobilization in ventricular myocytes

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It is well known that the integrity of the circulatory system can be influenced by changes in the flux of intracellular calcium ([Ca²⁺]_i) in heart. Ca²⁺ movements are closely related to cardiac electro-physiological events, membrane integrity, energy metabolism and contractile function. Maintaining the intracellular free Ca²⁺ concentration requires a fine balance in the co-ordinated function of many organelles. Intracellular organelles which incorporate Ca²⁺ from and return Ca²⁺ to the cytosol play a central role in determining [Ca²⁺]_i in the cell [15]. In a mammalian ventricular cell, Ca²⁺ moves across the sarcolemma (SL), sarcoplasmic reticulum (SR), and mitochondrial membranes with the help of L-type Ca²⁺ channels, Na–Ca exchangers, Ca²⁺-ATPase, and Ca²⁺ binding sites within the SL, Ca²⁺ release channels and Ca²⁺-ATPase within the SR and Na–Ca exchangers and Ca²⁺-binding proteins within the mitochondrial membranes. It is assumed that the cytosolic concentration [Ca²⁺]_i and spatial distribution of the [Ca²⁺]_i transients are uniform within the cell [16]. During cardiac excitation, extracellular Ca²⁺ moves into the cells through activated Ca²⁺ channels and reverse Na–Ca exchange [17, 18]. This Ca²⁺ current (I_Ca) may in turn control SR Ca²⁺ release via the SR Ca²⁺ release channel/ryanodine receptor [19]and contraction ensues. For relaxation to occur, Ca²⁺ must be removed from the cytoplasm, either by transport into the SR (by SR Ca²⁺-ATPase) or out of the cell (by sarcolemmal Ca²⁺-ATPase or Na–Ca exchange). These transport systems are thus in competition for cytoplasmic Ca²⁺ and act in concert, both to regulate the intracellular Ca²⁺ concentration and to modulate Ca²⁺-dependent events [15, 20].

	3 Intracellular Ca2+ in the diabetic heart

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Evidence for abnormal myocardial cell function in diabetes mellitus, influenced by metabolic changes, has appeared in recent years. Experimental studies at the cellular level have provided data for several possible explanations for this cardiac dysfunction. Among these, an abnormal intracellular Ca²⁺ homeostasis and trans-sarcolemmal receptor signalling defects have been suggested [5, 21]. For example, during chronic diabetes intracellular Ca²⁺ homeostasis in the heart is altered, possibly as a result of an impairment in SR which acts as a Ca²⁺ store and takes up and releases Ca²⁺ on a beat-to-beat basis [22]. Additionally, SL Ca²⁺ pump and Na–Ca exchange activity, together with the efflux of Ca²⁺ through the SL are depressed in the diabetic heart [23, 24]. Mitochondrial Ca²⁺ abnormalities have also been reported in the chronically diabetic rat heart [5]. Among the receptor defects, cardiac β-adrenoceptor function is altered in diabetic animals. These alterations include a depressed β-receptor number, a reduced response to β-adrenergic agonists, and a significant alteration in the response and sensitivity to perfusate Ca²⁺ stimulation [10, 25–27]. These results collectively support the hypothesis that diabetes mellitus leads to an alteration in Ca²⁺ movements in the heart.

In more recent years, intracellular Ca²⁺ mobilization and contractility in the diabetic heart have been studied using isolated cardiomyocytes that have the responses of an intact heart but are not affected by non-myocardial tissues [28]. As single cells, they are suitable for measuring contractility, ion activities and Ca²⁺ mobilization, and an increasing number of reports have documented intracellular Ca²⁺ transients and ion channel and transporter activities in parallel with single cell contraction in diabetic heart cells [29–34]. Utilization of fluorescent dyes in isolated myocytes allows for the analysis of [Ca²⁺]_i, and the study of the mechanism by which stimulation causes [Ca²⁺]_i changes. Moreover, the restoration of [Ca²⁺]_i following stimulation-induced elevation can be observed, especially with rapidly desensitizing agonists that produce a brief increase in Ca²⁺ in the cytosol [35–39]. Using these methods, it has been repeatedly reported that cardiomyocyte contraction is impaired in diabetic hearts [29, 31, 40]. Cell shortening and velocities of contraction (+dL/dt) and relaxation (–dL/dt) were significantly lower in diabetic myocytes than controls, whereas time to peak shortening was prolonged in diabetic cells [34]. These observations agree with the results from intact hearts and tissue preparations of diabetic rats which show decreased peak ventricular pressure development and rates of ventricular pressure development and decline (±dP/dt).

The data on basal levels of [Ca²⁺]_i in diabetic myocytes are more inconsistent. Early studies have reported an increase in resting [Ca²⁺]_i, an effect that appeared to support the hypothesis of Ca²⁺ overload in diabetic rat hearts [30, 41]. However, the animals used in the above studies were claimed to be a model of non-insulin-dependent diabetes, and different from STZ-diabetic rats, often referred to as a model of poorly controlled insulin-dependent diabetes. Subsequently, Noda et al. [31, 42]reported a reduced basal [Ca²⁺]_i in the diabetic heart and suggested a relationship between depressed basal [Ca²⁺]_i and reduced cardiac contractility. However, their measurements were made at an extracellular Ca²⁺ concentration of 0.5 mM that is not physiological for rats. In our laboratory, a large amount of data has been collected that demonstrates no differences in basal [Ca²⁺]_i levels between diabetic and control cardiomyocytes [32, 34, 40]. Hence, under normal conditions for isolated cardiomyocytes, basal [Ca²⁺]_i (both resting and electrically stimulated) does not necessarily reflect the depressed contraction in the diabetic heart and [Ca²⁺]_i transients in response to different physiological/pharmacological stimulants may be better parameters to represent cardiac dysfunction.

	4 [Ca2+] mobilization in the diabetic heart

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4.1 Sarcolemmal Ca²⁺ channels and Ca²⁺ and K⁺ currents in diabetic cardiomyocytes
Ca²⁺ channel activity in sarcolemmal membrane of diabetic hearts has been extensively studied. Unfortunately, the data are controversial. Increased PH200-110 (a dihydropyridine derivative) binding sites in diabetic cardiac SL membrane [43], and decreased nitrendipine binding sites with increased affinity were observed in diabetic cardiac membranes [44]. Net influx of Ca²⁺ was reported to be significantly reduced in chronic (4–8 weeks) diabetic rat myocardium [45]. L-type Ca²⁺ current was not significantly changed [46]. There are data indicating a prolongation of the action potentials in diabetic rat papillary muscles [47], and in isolated diabetic myocytes [29]perhaps due to enhanced Ca²⁺ current [47, 48]. Prolonged action potential duration in 30-week diabetic myocardium has also been reported in rat ventricular muscle and ventricular myocytes [49]. Whole cell clamp in cardiomyocytes revealed a considerable reduction of the transient outward current (I_to), but the inward rectifier potassium current (I_Ki) was found to be unaltered [49]. The diabetes-related suppression of I_to with accelerated inactivation explains the decreased repolarization rate of the action potential [49], and may act to force more Ca²⁺ to enter the myoplasm. Thus, the Ca²⁺ transient probably has an increased amplitude, and lasts longer. Due to this, the contractile response is prolonged (sometimes potentiated) in papillary muscles [4], and in isolated myocytes [29]. Under such conditions, [Ca²⁺]_i may be increased while the Ca²⁺ content of the SR is probably decreased. This situation can explain why the total Ca²⁺ content of isolated myocytes is smaller, and the effect of some inotropic agents is diminished [29]. The transient outward potassium current density was significantly reduced by diabetes whereas the voltage dependence of the inactivation and the time dependence of recovery were not modified [46]. Furthermore, the lengthening of the action potential induced by diabetes results mainly from a decrease of the transmembrane Ca²⁺-independent potassium permeability [46]. Insulin restored the density of the 4-aminopyridine-sensitive early transient component of I_to which decreased in diabetes [50]. The prolongation of APD suppresses the extrusion of Ca²⁺ via (electrogenic) Na–Ca exchange, and facilitates the sustained increase of the net Ca²⁺-influx [48].

The lack of effect of diabetes on I_ki [49], the main component of the resting conductance of cardiomyocytes, would account for the lack of change in resting potential of myocytes from diabetic rats as compared to controls. Decreased I_to in diabetic cells will mainly delay the early phase of repolarization of the action potential. I_to and the Ca²⁺ current (I_Ca) activate in the same range of potentials. One consequence of a depressed I_to during diabetes would be a longer Ca²⁺ influx through transmembrane Ca²⁺ channels. This could explain the greater reduction of APD induced by Ca²⁺ blockers in ventricular myocardium of diabetic rats [48]. Delayed rectifier current (I_K) is involved in the slower phase of action potential repolarization; decrease of this current in diabetic myocytes may contribute to the lengthening of the late repolarization phase of the action potential [46]. In a recent report, rate-dependent attenuation of I_K is more pronounced in cells from diabetic rats [51].

4.2 Na–Ca exchange activity in SL of diabetic myocardium
Depressed Na–Ca exchange activity without change in affinity to Ca²⁺ has been previously reported in diabetic rats [24]. These changes may be due to compositional modifications in the SL membrane, possibly as a result of diabetes-induced hyperlipidemia [7]. In this regard, unpublished observations from our laboratory have indicated that the Na–Ca exchange in hypertensive-diabetic rats demonstrates a more significant increase in the affinity for Ca²⁺. Interestingly, the hypertensive-diabetic rat model is known for its severe hyperlipidemia [7, 52].

4.3 SR Ca²⁺-ATPase in diabetic cardiomyocytes
It has been well documented that SR function in diabetic hearts is depressed. The early report of SR dysfunction in the diabetic rat heart came from isolated cardiac membrane vesicles whereby the uptake of ⁴⁵Ca²⁺ into SR vesicles (a function of SR Ca²⁺-ATPase) was reported to be reduced [22]. More recently, Ca²⁺ transients have been measured to elucidate SR function. In this regard, a depressed rapid-cooling contracture (1°C, an indirect measure of SR releasable Ca²⁺) was reported in papillary muscles [53]and isolated cardiomyocytes, accompanied by a reduction in intracellular Ca²⁺ transients [34]. Caffeine can also induce Ca²⁺ release from the SR, and the resulting contracture can be used as an indirect measure of SR Ca²⁺ available for release. Caffeine-induced Ca²⁺ transients and contracture are both depressed in diabetic myocytes [32, 34].

4.3.1 Rapid-cooling and caffeine contractures
The Ca²⁺ content of the SR that is available for release is clearly an important determinant of the contractile state. Two approaches have been used to study SR Ca²⁺ in cardiomyocytes: rapid application of (a) cold solution (RCC, 1°C) or (b) caffeine to induce SR Ca²⁺ release. The resulting contracture can be used as an index of SR Ca²⁺ available for release. The advantage of using the above approaches is that they can be done ‘on line’ with contracted living cells [15].

Rapid cooling of cardiac muscle results in a contracture that can be attributed to the quick release of SR Ca²⁺ to the cytoplasm. The amplitude of the contracture is indicative of the amount of Ca²⁺ available for release from SR at the time of cooling. During the cooling process, the probability of the SR Ca²⁺ release channel opening increases [54], the action potential duration is increased [55], peak Ca²⁺ current decreases, myofilament Ca²⁺ sensitivity is reduced [56], and Na–Ca exchange activity is attenuated [56]. The duration of [Ca²⁺]_i elevation during an RCC in unloaded conditions prolongs the active state and may allow the myocyte to shorten progressively. Due to inhibition of membrane Ca²⁺ transport (Ca²⁺ pump and Na–Ca exchange), cell shortening declines slowly and the rise in [Ca²⁺]_i during an RCC is less transient than that observed during caffeine-induced contractures. As [Ca²⁺]_i is much higher during an RCC than during a normal twitch, it appears that rapid cooling can release all of the available SR Ca²⁺ [15], while only a fraction of the SR Ca²⁺ available for release is discharged during a normal twitch.

Caffeine increases SR Ca²⁺ channel opening and hence promotes a Ca²⁺ leak into the myoplasm. This process effectively prevents the SR from accumulating Ca²⁺. Ca²⁺ pumped into the SR is immediately reintroduced into the myoplasm via the open SR channels, and can then be removed from the cell by Na–Ca exchange. Hence, caffeine can inhibit SR Ca²⁺ uptake without directly effecting SR Ca²⁺ pump. One caveat is that caffeine can cause myofilament sensitization and phosphodiesterase inhibition that can increase cAMP and cAMP-dependent protein kinase and both these effects can complicate interpretation of the results [56]. Moreover, Indo-1 fluorescence is strongly quenched by caffeine. However, this occurs in a wavelength-independent manner so that the fluorescence ratio used to estimate [Ca²⁺]_i is unaffected.

4.3.2 Caffeine-induced calcium transients in quiescent myocytes
The SR content of Ca²⁺ can also be assessed by measuring [Ca²⁺]_i response to caffeine with the fluorescence Ca²⁺-indicator, fura-2. Brief exposure to caffeine induces a transient inward current which reflects the electrogenic extrusion of Ca²⁺ across the membrane by the Na–Ca exchange [57]. With this method, it was demonstrated that the peak [Ca²⁺]_i transient in response to caffeine was significantly decreased in diabetes, suggesting a reduction in Ca²⁺ storage in the SR [34]. Insulin treatment prevented this effect [34]and this was probably related to the fact that insulin treatment of STZ diabetic rats reverses the depression of Ca-ATPase activity and Ca²⁺ uptake by the SR [22].

In diabetic hearts, there has been some debate as to whether Ca²⁺ overload or Ca²⁺ underload occurs, and whether the decrease in cardiac contractility in the diabetic rat is accompanied by reduced or excessive loading of the Ca²⁺ into SR. Our results, documenting the decline in SR Ca²⁺ store and release, as assessed by RCC and caffeine-induced [Ca²⁺]_i transients [34], agree with the results of Bouchard and Bose [53]who reported a reduction in SR Ca²⁺ stores and decreased fractional release of Ca²⁺ during stimulation of papillary muscles from STZ-treated rats. Thus, the marked reduction of developed tension in diabetic tissues is suggested to be a consequence of depleted SR Ca²⁺ stores, rather than a result of chronic SR Ca²⁺ overloading. In this regard, the reduced Ca²⁺ uptake by the SR [22]could diminish Ca²⁺ stores and hence impair Ca²⁺ release, with a consequent reduction in cardiac contraction.

4.4 Mitochondrial function in diabetic hearts
The current view of Ca²⁺ accumulation by heart mitochondria is that it may act as a sink for Ca²⁺ under pathological conditions. Therefore, mitochondria may act as a potent and important buffering component of Ca²⁺ in the heart. A generalized depression of mitochondrial function exists in hearts from diabetic rats. Mitochondrial Ca-ATPase activity and Ca²⁺ transport capacity are significantly impaired in hearts from chronically diabetic rats [58]. Although the alteration in Ca²⁺ accumulation by mitochondria may have little effect on cardiac function on a beat-to-beat basis, any stimulus to the heart that results in elevated [Ca²⁺]_i may not be adequately buffered in diabetic hearts. Direct measurement of mitochondrial calcium concentration in diabetic cardiomyocytes has not been made, although a technique for such a measurement is available and has been applied in other pathological models such as anoxia and reoxygenation [59, 60]. In a report using isolated cardiomyocytes, mitochondrial uptake of ⁴⁵Ca²⁺, transmembrane potential gradient across the inner membrane of mitochondria, and cell respiration were significantly decreased in diabetic myocytes compared to control [61]. These changes were restored to normal by insulin treatment [61].

4.5 Myofilaments and Ca²⁺ sensitivity in diabetic cardiomyocytes
Two kinds of Ca²⁺-dependent alterations may affect contractility of the diabetic heart: (a) changing the availability of Ca²⁺ to the myofilaments, and (b) modifying the responsiveness of the myofilaments to activation by intracellular Ca²⁺. The availability of intracellular Ca²⁺ is regulated by the SL and SR, and Ca²⁺ responsiveness is controlled by the myofilaments and the regulatory troponin–tropomyosin complex [62].

There has been no consistent finding regarding the sensitivity of the myofilament to Ca²⁺. The sensitivity of isolated myofibrils to [Ca²⁺]_i has been reported to be unchanged [63]or increased [64]during diabetes. The increased sensitivity has been suggested to contribute to the slow time-course of relaxation. More recently, two studies have suggested that the sensitivity of myofilaments to Ca²⁺ was decreased during diabetes [65, 66]. Although these experiments were performed under different conditions, the data appear to suggest that altered intracellular Ca²⁺ transients are not the only cause of cardiac dysfunction during diabetes.

It has been reported that the lower activity of myosin ATPase and abnormal myosin isoenzyme distribution could be responsible for the depressed contractile function in diabetic myocardium [67]. Additionally, Ca²⁺-sensitivity of skinned diabetic myocytes is significantly enhanced with unchanged maximal activated tension [64]. In contrast, increased expression in cardiac β-myosin heavy chain and changes in troponin T expression may contribute to the decrease in Ca²⁺-sensitivity of myofilaments at pH 7.0, and decreased maximum tension-generating ability at pH 6.6 in diabetes [66]. A decreased Ca²⁺-sensitivity of isometric tension in skinned cardiomyocytes from diabetic rats suggests that decreased cardiac output in the whole heart can occur independently of alterations in Ca²⁺-handling by the SR or SL. During in vivo acidotic conditions such as ischemia and hypoxia, a substantial decrease in ventricular pressure may occur, in part due to changes in myofilament protein expression [66]. The apparent Ca²⁺-sensitivity was greatly diminished at a sarcomere length of 1.9 µm but was not affected at a longer length (2.4 µm). Another possibility is that diabetic Ca²⁺-sensitivity changes in the myocardium are coupled with troponin T alterations [65].

4.6 [Ca²⁺]_i responses to β-adrenergic stimulation
Diabetic rat hearts are characterized by diminished responses to β-adrenergic stimulation [10, 68]. This has been suggested to be due to a reduction in β-adrenergic receptor [25]. As electrically stimulated diabetic cardiomyocytes demonstrate a depressed maximum [Ca²⁺]_i response to isoproterenol and 8-bromo-cAMP without a change in sensitivity [31, 40], it suggests that in addition to alterations in β-adrenoceptor function there are postreceptor defects in the diabetic myocardium. The response of [Ca²⁺]_i to isoproterenol can be blocked by thapsigargin, suggesting that the β-agonist-induced [Ca²⁺]_i changes are mediated, in part, by SR Ca²⁺-ATPase [40]. Insulin treatment of diabetic rats reversed the depressed response of [Ca²⁺]_i to isoproterenol [40].

4.7 [Ca²⁺]_i responses to different pharmacological agents
As stated earlier, the peak [Ca²⁺]_i transient induced by caffeine may be used as an indirect index of SR releasable Ca²⁺. The maximum [Ca²⁺]_i increase in response to ouabain is reduced in diabetic cells while the sensitivity of diabetic myocytes to ouabain was enhanced [32]. KCl-induced [Ca²⁺]_i increase was enhanced in diabetic cells accompanied by a decreased caffeine and dichlorobenzamil blockade of the KCl effects [32]. Nitrendipine blockade effects were similar in diabetic and control cells [30, 32]. The maximum responses of [Ca²⁺]_i to exogenous ATP was increased in diabetic cells [32].

4.7.1 Isoproterenol and 8-bromo-cAMP
In addition to the depressed response to isoproterenol, diabetic myocytes also demonstrate an attenuated [Ca²⁺]_i response to 8-bromo-cAMP at 10^–5 M and higher concentration [32]. This suggests that at steps distal to the β-adrenoceptor and adenylate cyclase, diabetic myocytes exhibit a deficiency. Hence, there may be an alteration between cAMP and [Ca²⁺]_i increase such as PKA activation and the phosphorylation of proteins. The phosphorylation process in the SR of diabetic myocytes is as yet unclear.

4.7.2 Ouabain
The response to ouabain in papillary muscle and left atria from hearts of diabetic rats was reported to be markedly depressed [69, 70]. It was also reported that the maximum number of high- and low-affinity ouabain binding sites in membrane preparations obtained from chronically diabetic rats was significantly reduced to 60 and 49% of controls, respectively. These results suggest that the decreased inotropic response of ouabain in the intact cardiac tissue obtained from diabetic rats may be related to a decreased number of ouabain binding sites [71]. The altered K_d could be due to an alteration in the ouabain binding sites or an altered composition of the membrane in diabetes. The activity of Na-K ATPase is dramatically reduced in the diabetic heart [72]. As this enzyme indirectly regulates [Ca²⁺]_i levels through its modulation of [Na⁺]_i, the diabetes-linked decrease in Na-K ATPase activity would be expected to mediate a net increase in both [Na⁺]_i and [Ca²⁺]_i. Myocardium from diabetic rats is susceptible to Ca²⁺ loading by ouabain incubation (as measured by afterdepolarizations) [73]. These data suggest a decline in the reserve capacity of the sarcolemmal Na⁺ pump in the diabetic heart. The decrease in Na-K ATPase could enhance the sensitivity to digitalis-like compounds by reducing the reserve capacity of the Na-pump and hence, the extent of digitalis-induced pump inhibition required before the onset of toxicity (in the case of single myocyte, hypercontraction). A reduction in reserve capacity may lower the tolerance to ouabain by decreasing the number of pump sites that the glycoside would have to inhibit before eliciting a marked [Na⁺]_i accumulation and the resulting toxic effects believed to be mediated by Ca²⁺-overload. Cardiac arrhythmias are a frequent and serious complication of the clinical use of digitalis glycosides, and it is possible that the tolerance to these cardiotoxic effects and the margin of safety for cardiotonic steroids is reduced in diabetic patients.

4.7.3 KCl and ATP
The influx of [Ca²⁺]_i is a prerequisite of the KCl-induced [Ca²⁺]_i transient because EGTA abolished the transient. The [Ca²⁺]_i influx is triggered by membrane depolarization and thus activation of L-type Ca²⁺-channels and ‘reversed’ mode operation of Na–Ca exchange. Ca²⁺ influx via Ca²⁺-channel and Na–Ca exchange may directly contribute to the [Ca²⁺]_i increase and, more importantly, induce Ca²⁺ release from SR. In accordance with our results, an enhanced [Ca²⁺]_i response to KCl (30 mM) in a myocyte suspension from diabetic rats has been documented [30]. Increased L-type Ca²⁺-channel activity determined by [³H]PN-200-110 binding sites in cardiac membrane has been reported in the diabetic heart [43]. Interestingly, the [³H]PN-200-110 binding to control cardiac membrane was dose-dependently inhibited by verapamil, but this was not the case in diabetic cardiac membranes [43]. This suggests that L-type Ca²⁺-channels are quantitatively and qualitatively altered in diabetes, and may be related to the nitrendipine effects found in this study. The effects of ATP in modulating Ca²⁺-channel and intracellular Ca²⁺ store are very similar to KCl [74]. They are both sensitive to BAY K 8644, nifedipine, and EGTA and both are partially inhibited by ryanodine and caffeine. These similarities suggest that the ATP mechanism is similar to the KCl effects of membrane depolarization [74]and direct activation of Ca²⁺-channels.

	5 Summary

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The quality control indices of myocyte isolation (viability, yield, survival time, cell response, etc.) suggest that the adult rat myocyte model is stable and useful in [Ca²⁺]_i measurements and functional studies at the cellular level. Moreover, diabetic cardiomyocytes are a valuable model for studying cellular functions of the diabetic heart as they retain most of the features of cardiac dysfunction of intact rat. Data from our studies indicate that the basal [Ca²⁺]_i in both quiescent and electrically-stimulated cells is not changed. Thus, resting levels of [Ca²⁺]_i and basal [Ca²⁺]_i transients may not reflect the abnormalities observed in diabetes until the system is challenged by certain stimuli.

[Ca²⁺]_i responses to isoproterenol are depressed in both resting and stimulated diabetic cells. This suggests an alteration in the β-adrenergic pathway, possibly related to the β-adrenoceptor deficiency reported in the diabetic heart. SR Ca-ATPase is also involved in the isoproterenol-induced [Ca²⁺]_i changes. Moreover, the decreased maximum response to 8-bromo-cAMP provides evidence of a post-receptor alteration in the pathway. Diabetic myocytes are more sensitive to ouabain, whereas the maximum response to ouabain was depressed. This may be the result of depressed Na-K ATPase and increased [Na⁺]_i.

In diabetic myocytes, rapid cooling contractures and caffeine contractures are depressed, whereas caffeine-induced Ca²⁺ transients are decreased. Ryanodine binding suggests a decreased number of high-affinity binding sites in the SR of diabetic myocytes. Additionally, there are indications that SR releasable calcium is reduced and that the major functions of SR, notably uptake, release and storage, may be depressed in diabetic myocytes. Finally, L-type Ca²⁺-channels are quantitatively and qualitatively altered in diabetes. Insulin treatment normalizes most of the diabetes-induced changes in cardiomyocytes, suggesting that metabolic alterations due to insulin deficiency play an important role in diabetic cardiomyopathy.

Results from several studies show that in diabetes the function of major organelles which handle [Ca²⁺]_i in myocytes is depressed, which in turn causes the alteration of [Ca²⁺]_i mobilization in myocytes. Different second messenger systems involved in E-C coupling may also be altered due to the metabolic impairments. The rapid increase in our understanding of the pathophysiology of calcium homeostasis in cardiomyocytes will be forthcoming as the powerful new tools of molecular and structural biology are used to investigate the regulation of the Ca²⁺ transport system.

Time for primary review 33 days.

	Acknowledgements

 
Some of the studies described in this paper were supported by an operating grant from the Heart and Stroke Foundation of B.C. and Yukon. The financial support of the Canadian Diabetes Association (Scholarship to B. Rodrigues) and the Canadian Heart and Stroke Foundation (Fellowship to J.Z. Yu) is gratefully acknowledged.

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