Calcium Signaling in the Ventricular Myocardium of the Goto-Kakizaki Type 2 Diabetic Rat

The association between diabetes mellitus (DM) and high mortality linked to cardiovascular disease (CVD) is a major concern worldwide. Clinical and preclinical studies have demonstrated a variety of diastolic and systolic dysfunctions in patients with type 2 diabetes mellitus (T2DM) with the severity of abnormalities depending on the patients' age and duration of diabetes. The cellular basis of hemodynamic dysfunction in a type 2 diabetic heart is still not well understood. The aim of this review is to evaluate our current understanding of contractile dysfunction and disturbances of Ca2+ transport in the Goto-Kakizaki (GK) diabetic rat heart. The GK rat is a widely used nonobese, nonhypertensive genetic model of T2DM which is characterized by insulin resistance, elevated blood glucose, alterations in blood lipid profile, and cardiac dysfunction.


Use of the Goto-Kakizaki Diabetic Rat
Diabetes mellitus (DM) is a metabolic disease characterized by abnormal glucose homeostasis and defects in insulin metabolism. Cardiovascular disease (CVD) is the leading cause of death in the diabetic population. However, the molecular mechanisms underlying diabetic cardiomyopathy remain unclear.
Animal models are increasingly being used to elucidate the mechanisms underlying diabetic cardiomyopathy in both type 1 and type 2 diabetes. One of the most widely used animal models of type 2 diabetes mellitus (T2DM) is the Goto-Kakizaki (GK) rat. The GK rat is a polygenic nonobese model of T2DM. This model is generated by selective inbreeding of mildly glucose-intolerant Wistar rats over many generations [1]. At least 17 genes associated with metabolism, signal transduction, receptors, and secreted factors are involved in the pathogenesis of diabetes in the GK rat [2]. The general characteristics of the GK rat include fasting hyperglycemia, impaired insulin secretion in response to glucose both in vivo and in isolated pancreata, raised glycosylated hemoglobin, hepatic and peripheral insulin resistance, altered heart and body weight, and a variety of late complications, including cardiomyopathy, nephropathy, and neuropathy [1,[3][4][5][6][7][8][9][10][11]. In contrast to many other non-insulin-dependent rodent models, GK rats are non-obese [1,12].
Three genetic loci are responsible for coding and transferring diabetic pathology to the fetus, and these include genes that are responsible for a reduction in β-cell mass and reduced insulin secretion [12]. During the prediabetic period (first three weeks after birth), animals have reduced body weight and do not show hyperglycemia. After weaning, many changes occur which include hyperglycemia, impaired glucose-induced insulin secretion (due to defective prenatal β-cell proliferation and reduction in β-cell mass), reduced insulin sensitivity in the liver, and moderate insulin resistance in peripheral tissues [12,13]. Persistent hyperglycemia over time provokes pancreatic islet inflammation, oxidative stress, fibrosis, and finally β-cell dysfunction. In fact, the pancreatic islets of adult GK rats show decreased β-cell number and insulin content as compared to their age-matched control animals [12].
GK rats have been considered as one of the best nonobese type 2 diabetic animal models. GK rats exhibit valuable characteristics that are more or less common and functionally present in human diabetic patients. This animal model is considered appropriate to examine various pathologic mechanisms of T2DM [12,14]. As mentioned earlier, reduced β-cell mass and reduced β-cell function are key characteristics found in this animal model [15]. Therefore, it is clear that GK rats form an important resource in preclinical T2DM research [16] in order to study the role of β-cell compensation in the pathogenesis of T2DM.
An earlier study has shown that GK islet fibrosis is accompanied by marked inflammation which is a characteristic that has been reported in islets of type 2 diabetic patients [17]. Other changes that are common between GK rats and human diabetic patients include decreased activity of glucose transporter (GLUT-2), glycerol-3-phosphate dehydrogenase (GPDH), and glucokinase and changes in the lipid profile [12].
As in humans, GK rats also develop renal lesions, structural changes in peripheral nerves, and retinal damage [13]. For example, in adult GK rats, significant morphological alterations in kidneys occur in response to chronic hyperglycemia which are similar to that in human diabetic patients [18,19]. These morphological changes in kidneys include glomerulosclerosis, proliferation of mesangial cells, atrophy of basement membrane, and tubulointerstitial fibrosis [20].

Other Animal Models of Type 2 Diabetes
T2DM is characterized by insulin resistance and the inability of the β-cell to sufficiently compensate, which leads to hyperglycemia [21]. In addition, T2DM is closely associated with obesity which is one of the main pathological causes of insulin resistance [15,22]. Many animal models are therefore obese as a result of naturally occurring mutations or genetic manipulation and are useful in understanding obesity-induced insulin resistance and its effects. These are divided into monogenic models, polygenic models, and diet-induced models [23]. The general characteristics for these obese models are insulin resistance and impaired glucose tolerance. In other words, these models lack sufficient insulin secretion required to compensate for the insulin resistance as part of the obesity (obesity-induced hyperglycemia) [13,23].
Lep ob/ob mice, Lepr db/db mice, and Zucker diabetic fatty rats are the most commonly used models of monogenic obesity. They have a disrupted leptin signaling pathway, leading to hyperphagia and obesity [13]. Polygenetic animal models, however, provide more accurate models of the human condition [15]. These include KK-A Y mice, New Zealand obese (NZO) mice, TallHo/Jng mice, and Otsuka Long Evans Tokushima Fat (OLETF) rat. Obesity can also be induced by feeding the rodent a high-fat diet (diet-induced models). The weight gain in these animals is associated with insulin resistance and abnormal glucose metabolism [12,13,23].
In contrast to the animal models mentioned above, the GK rat is a non-obese animal model of T2DM. It is characterized by reduced β-cell mass and/or β-cell function [24]. The GK rat is glucose intolerant and displays defective glucoseinduced insulin secretion. Furthermore, the development of insulin resistance does not seem to be the main initiator of hyperglycemia. Instead, the defective glucose metabolism is regarded to be due to reduced β-cell mass [25] and/or function [26]. Adult GK rats show a 60% decrease in their total pancreatic β-cell mass. Blood glucose is elevated only after the first 3-4 weeks of animal's age, and blood glucose rises significantly after a glucose challenge [13,27]. The GK model is characterized by early hyperglycemia, hyperinsulinemia, and insulin resistance, [1,12]. Other examples of non-obese animal models of T2DM are the C57BL/6 (Akita) mutant mouse, the Cohen diabetic rats, and the spontaneously diabetic Torri (SDT) rats [13].

Blood Chemistry in the Goto-Kakizaki Diabetic Rat
Blood insulin, glucose, and lipid profiles in the GK rats compared to controls are summarized in Tables 1, 2, and 3, respectively. Blood insulin is either unaltered [28][29][30][31][32][33][34] or increased [29,34,35] in the GK rats (Table 1). Fasting blood glucose and nonfasting blood glucose are slightly increased [10,11, and urine glucose is increased [30] in the GK rat. Following a glucose challenge, in the fasted state, blood glucose is significantly elevated at 30, 60, and 120 min [29, 37-40, 44, 46, 48-50] in the GK rat indicating end organ resistance to the action of insulin (Table 2). Blood cholesterol is increased [29,35,43,44] whilst high-density lipoprotein cholesterol may be either unaltered [31] or increased [44] and low-density lipoprotein cholesterol is unaltered [31,44] in the GK rat compared to controls. Blood free fatty acids are either unaltered [11,31] or increased [38,45] in the GK rats compared to controls. Triglycerides are either increased [38,[43][44][45] or unaltered [2,30,45] in the GK rats compared to controls ( Table 3). Part of the variability in blood chemistry may be attributed to the age of the animals and dietary regime. In summary, the GK rat displays hyperglycemia, insulin resistance, and disturbances in lipid profile.
Earlier studies have reported that chronic mild hyperglycemia produces molecular and structural correlates of hypertrophic myopathy in GK rats [40]. Several mechanisms whereby hyperglycemia may induce left ventricle remodeling have been proposed. One of these mechanisms is the increased activity of profibrotic and prohypertrophic cytokine transforming growth factor-β1 (TGF-β1) in the ventricular tissue [52]. TGF-β1 reproduces most of the hallmarks seen in structural remodeling. Specifically, TGF-β1 induces expression levels of extracellular matrix (ECM) constituents by cardiac fibroblasts (i.e., fibrillar collagen, fibronectin, and proteoglycans), self-amplifies its own expression in both cardiac myocytes and fibroblast [53,54], and stimulates the proliferation of fibroblasts and their phenotypic conversion to myofibroblasts [55,56]. D'Souza et al. have shown that the increased activity of TGF-β1 and phosphorylation of protein kinase B (PKB)/Akt and its downstream effectors mediate the hypertrophic effects of TGF-β1 in the prediabetic GK left ventricle [36]. The hypertrophic events were also sustained in the aging GK myocardium [40]. Earlier studies have suggested that enhanced activity of myocardial Na + /H + exchanger plays a role in the molecular mechanisms involved in cardiac hypertrophy. It is likely that the activation of the Akt pathway mediates the hypertrophic effect of myocardial Na + /H + exchanger in the GK rat model of T2DM [28]. Interestingly, several studies have shown that female rat hearts are more hypertrophied than male hearts [10,32,57].

Hemodynamic Function in the Isolated Perfused Goto-Kakizaki Rat Heart
Heart rate in the isolated perfused heart is lower in comparison to the heart rate in vivo in GK and control hearts (Table 7). Isolated perfused heart rate is unaltered [10,11,31,50] in GK rats. Left ventricle +dP/dt and -dP/dt are either unaltered [10,31,59] or reduced [51] in the GK rat. Coronary flow is either reduced [11,31] or unaltered [10] in GK rats compared to controls. Collectively, the GK rat heart displays a variety of abnormal hemodynamic characteristics, including altered rate of development and relaxation of ventricular contraction and altered coronary flow compared to controls.

Contraction in Ventricular Myocytes from the Goto-Kakizaki Rat Heart
Characteristics of shortening in myocytes from GK rats compared to controls are shown in Table 8. Myocyte diameter, surface area, cross-sectional area, and cell capacitance were increased [28,30,33,36,40,51], and resting cell length may be unaltered [10,39,41,50] or increased [47] in myocytes from the GK rat. In electrically stimulated myocytes, the time-to-peak (TPK) shortening was prolonged HOMA-IR 7, 11, and 15 w Increased at 7 * , 11 * , and NSD 15 (w) [29] FBG: fasting blood glucose; NFBG: nonfasting blood glucose; UG: urine glucose; OGTT: oral glucose tolerance test; HbA1c: glycated hemoglobin A1c; HOMA-IR: homeostasis model assessment-estimated insulin resistance; NSD: no significant difference. * Significant difference. [39,41,47] or unaltered [48,50] and the time-tohalf (THALF) relaxation of shortening may be unaltered [41,47,48] or shortened [50] or lengthened [39] in myocytes from the GK rat. Amplitude of shortening may be unaltered [10,41,48,50] or increased [39] in myocytes from the GK rat. In summary, ventricular myocytes from the GK rat heart tend to be larger in size and have prolonged time course and generally similar amplitude of contraction compared to myocytes from the control heart. During the process of excitation-contraction coupling (ECC), the arrival of an action potential causes depolarization of the cardiac myocyte plasma membrane. This depolarization opens voltage-gated L-type Ca 2+ channels in the plasma membrane. The entry of small amounts of Ca 2+ through these channels triggers a large release of Ca 2+ from the sarcoplasmic reticulum (SR) via activation of the ryanodine receptor (RyR), by the process termed calciuminduced calcium release (CICR). The transient rise in intracellular Ca 2+ (Ca 2+ transient) results in the binding of Ca 2+ to troponin C which initiates and regulates the process of cardiac muscle cell contraction. During the process of relaxation, Ca 2+ is pumped back into the SR via the SR Ca 2+ -ATPase (SERCA2) and extruded from the cell, primarily via the Na + /Ca 2+ exchanger (NCX) [60,61]. Changes in the kinetics of shortening observed in myocytes of GK rats may be attributed, at least in part, to alternations in ventricular myocardial stiffness. Earlier studies have demonstrated increased collagen deposition and increased ventricular stiffness in different experimental models of T2DM, which in turn were associated with altered kinetics of myocardial contraction [62,63]. The observed disturbance in myocyte shortening may also be attributed to the alternation in the profile of expression of mRNA encoding various proteins involved in excitationcontraction coupling [48].

Intracellular Ca 2+ in Ventricular Myocytes from the Goto-Kakizaki Rat Heart
Characteristics of intracellular Ca 2+ in myocytes from GK rats compared to controls are shown in Table 9. Resting intracellular Ca 2+ is unaltered [10,41,47,48] or increased [28]; TPK Ca 2+ transient is unaltered [39,41,48,50] or prolonged [47]; THALF decay of the Ca 2+ transient is unaltered [39,47,48,50] or shortened [41]; and the amplitude of the Ca 2+ transient is unaltered [10,41,48], increased [47,50], or decreased [39] in myocytes from the GK rat. In wholecell patch clamp experiments, the amplitude, inactivation, and restitution of L-type Ca 2+ current are unaltered [48] in myocytes from GK rats compared to controls. Since intracellular Ca 2+ in cardiac cells is maintained by Ca 2+ influx (through L-type Ca 2+ channels; the primary trigger for SR Ca 2+ release) and efflux (through NCX; the major pathway for Ca 2+ efflux from the cell) [64], as well as Ca 2+ release (via the ryanodine receptors) and uptake by both SR (through SERCA2) and mitochondria, it is possible that the observed differences in these results may be attributed to differential changes in Ca 2+ transport activities in these organelles. Furthermore, the observed alterations in intracellular Ca 2+ may also be due to differences in the stage and severity of diabetes [65,66]. It is well known that alterations in SR Ca 2+ uptake and release mechanisms would impair cardiac cell function. Several studies have reported changes in cardiac SR Ca 2+ transport during the development of chronic diabetes [67][68][69][70][71]. For example, Ganguly et al. reported that a decrease in Ca 2+ uptake activity by SR was associated with a decrease in SERCA2a activity [68]. Furthermore, Golfman et al. showed that SR ATP-dependent Ca 2+ uptake activity was markedly decreased in the diabetic rat heart [72]. Yu et al. reported a reduction in both SR Ca 2+ content and ryanodine binding sites in diabetic hearts, indicating that the SR functions of storage and release of Ca 2+ were depressed [73]. It should be noted that prolonged depression of the SR Ca 2+ uptake activity in chronic diabetes may contribute to the occurrence of intracellular Ca 2+ overload [65].
In our recently published data, L-type Ca 2+ current and Ca 2+ transients were simultaneously measured in endocardial (ENDO) and epicardial (EPI) myocytes from the left ventricle of GK rats [74]. Consistent with previous findings [48], the amplitude of L-type Ca 2+ current, over a wide range of test potentials, was unaltered in ENDO and EPI myocytes from the left ventricle of GK rat. However, the amplitude of the Ca 2+ transients was reduced and by similar extents, in ENDO and EPI myocytes from the GK rat heart. The THALF decay of the Ca 2+ transients was reduced in EPI and ENDO myocytes from GK rats compared to controls. Interestingly, while a reduction in the amplitude of L-type Ca + current has been reported in earlier studies on a diabetic heart [75,76], it does not necessarily explain the reduced Ca 2+ transients. This is because many reports show no change in L-type Ca 2+ current despite the reduction in both contractions and Ca 2+ transients [48,74,[77][78][79]. Instead, reduction of Ca 2+ transients and the consequent contractile dysfunction may be due to depletion of SR Ca 2+ , which may result from RYR-dependent Ca 2+ leak, an increased Ca 2+ extrusion through NCX, or a reduced function of SERCA [61,80]. Further experiments will be required to investigate the role of SR in Ca 2+ transport in myocytes from the GK rat. Sheikh et al. [81]   receptor) activity [87,95] suggesting decreased SR Ca 2+ content, Ca 2+ uptake, and Ca 2+ release mechanisms in ventricular myocytes from the streptozotocin-induced diabetic rat.
Under pathological conditions, such as chronic diabetes, the mitochondria are able to accumulate large amounts of Ca 2+ , which serves as a protective mechanism during cardiac dysfunction and intracellular Ca 2+ overload. Therefore, altered mitochondrial uptake of Ca 2+ during diabetes may contribute to the reported decreased Ca 2+ transients. Although the mitochondria contribute to Ca 2+ signaling, their exact role in diabetic cardiomyopathy remains to be investigated.
Recent investigations, using animal models, suggest that mitochondrial dysfunction may also play a critical role in the pathogenesis of diabetic cardiomyopathy [65,71]. Potential mechanisms that contribute to mitochondrial impairment in diabetes include altered energy metabolism [96][97][98][99] oxidative stress [100][101][102], altered mitochondrial dynamics and biogenesis [103,104], cell death [105,106], and impaired mitochondrial Ca 2+ handling [107,108].  It should be noted that the main function of the mitochondria in the heart is to produce energy in the form of ATP, which is required for cardiac contractile activity. However, mitochondria are known to serve as Ca 2+ sinks in the cell by acting as a local buffering system, removing Ca 2+ and modulating cytosolic Ca 2+ concentrations [65,109]. In addition to controlling their intraorganelle Ca 2+ concentration, mitochondria dynamically interact with the cytosol and intracellular Ca 2+ handling machineries to shape the cellular Ca 2+ signaling network [65]. Recent evidence suggests that there is a dynamic exchange of Ca 2+ between the mitochondria and the cytosol and that mitochondrial Ca 2+ uptake increases mitochondrial ATP production [110]. Therefore, mitochondria can play an important role in preventing and/or delaying the occurrence of intracellular Ca 2+ overload in cardiomyocytes under   [65,71]. However, when the intramitochondrial Ca 2+ concentration exceeds its buffering capacity, irreversible swelling occurs leading to mitochondrial dysfunction. As a result, energy production as well as energy stores are depleted. Collectively, these defects may contribute to the development of cardiac dysfunction in diabetic cardiomyopathy [109]. Evidence of deficits in mitochondrial Ca 2+ handling has been demonstrated in animal models of both type 1 and type 2 diabetes. For example, in streptozotocin-(STZ-) induced diabetic rats, hyperglycemia was associated with lower rates of mitochondrial Ca 2+ uptake [107]. This reduction can be explained by the increased opening of the mitochondrial permeability transition pore (MPTP), resulting in the release of accumulated Ca 2+ . In STZinduced diabetic rats, Oliveira et al. observed that Ca 2+ uptake was similar in control versus diabetic hearts; however, mitochondria in diabetic hearts were unable to retain the accumulated Ca 2+ . This effect was not observed in the presence of cyclosporin, an MPTP inhibitor [108]. In type 2 diabetic ob/ob mice, reduced intracellular Ca 2+ release upon electrical stimulation, slowed intracellular Ca 2+ decay rate, and impaired mitochondrial Ca 2+ handling were observed [111,112]. Similarly, Belke et al. observed a reduction in Ca 2+ levels and a reduction in the rate of Ca 2+ decay in isolated cardiomyocytes from db/db animals, suggesting impaired mitochondrial Ca 2+ uptake [113]. Taken together, these studies support the notion that mitochondrial Ca 2+ handling is impaired in diabetic myocardium, resulting in compromised energy metabolism and thus reduced contractility.

Conclusion
Although diabetic cardiomyopathy is a frequent and important complication of DM, its physiological bases are still not completely understood. The GK type 2 diabetic heart displays a variety of abnormal hemodynamic characteristics in vivo and in the isolated perfused heart. Hyperglycemia is usually associated with alterations in heart rate, blood pressure, blood pumping capability, and/or coronary blood flow. Contractile function, in terms of amplitude and kinetics of shortening, is frequently disturbed in the GK type 2 diabetic heart. Several mechanisms may contribute to cardiac dysfunction including mitochondrial dysfunction, myocardial fibrosis, hypertrophy, and apoptosis. Many studies show no change in L-type Ca 2+ current despite the reduction in both contractions and Ca 2+ transient. Instead, reduction of Ca 2+ transients and the consequent contractile dysfunction may be attributed to both depletion of SR Ca 2+ , which may result from RyR-dependent Ca 2+ leak, an increased Ca 2+ extrusion through NCX, or a reduced function of SERCA (Figure 1). Understanding the molecular mechanism(s) of altered Ca 2+ signaling will provide opportunities for the development of new treatments to improve heart function in T2DM patients. Excitation-contraction coupling SR: