Diabetes mellitus (DM) is a major global health disorder currently affecting 450 million people. Diabetic cardiomyopathy (DC) is a disorder of cardiac muscle that is independent of coronary artery disease and that may lead to heart failure in diabetic patients. The precise mechanism(s) of diabetic cardiomyopathy are still not fully understood. Therefore, it is of paramount importance to develop experimental models of DM to study the time course and cellular, subcellular and molecular mechanisms of diabetic cardiomyopathy. With this in mind, scientists initially discovered that the antibiotic, streptozotocin (STZ) could be used to rapidly to induce diabetes mellitus in animal models. STZ destroys pancreatic beta cells, leading to hypoinsulinemia and hyperglycaemia. If left untreated hyperglycaemia may lead to DC and eventually heart failure. Initially in DM, the cardiac myocytes become apoptotic, disorganised and the number of myocytes are significantly reduced. The heart responds by enlarging itself (hypertrophy) which is accompanied by fibrosis leading to a physiological remodelling process. Within the myocytes, the process of excitation-contraction coupling (ECC) is deranged. This is due to an inability of the heart cells to regulate Ca2+ which is the initiator and regulator of cardiac muscle contraction. As a result, the heart takes longer to contract and to relax leading to DC, progressive heart failure and eventually sudden cardiac death. The aim of this review was to evaluate our current understanding of contractile dysfunction and disturbances in Ca2+ transport in the STZ-induced diabetic rat heart

 • Streptozotocin • Diabetes mellitus • Rat • Cardiomyopathy • Heart • Ventricle • Calcium signaling • Contraction

: Howarth FC, AlKury L, Smail M, Qureshi MA, Sydorenko V, et al. (2017) Effect of Streptozotocin-Induced Type 1 Diabetes Mellitus on Contraction and Calcium Tran

DM: Diabetes Mellitus; DC: Diabetic Cardiomyiopathy; STZ: Streptozotocin; ECC: Excitation-Contraction Coupling; SR: Sarcoplasmic Reticulum; SERCA2: Sarcoplasmic Reticulum Ca2+ ATPase Pump; TPK: Time to Peak; THALF: Time to Half; EPI: Epicardial; ENDO: Endocardial; DNA: Deoxyribonucleic Acid; mRNA: Messenger Ribonucleic Acid; RYR: Ryanodine Receptor

 The STZ-induced diabetic rat is a widely used experimental model of DM. STZ selectively enters pancreatic beta cells via the GLUT2 glucose transporter where it causes damage to the genetic machinery and ultimately leads to beta cell death [1]. The general characteristics of the STZ-induced diabetic rat include hypoinsulinemia, hyperglycemia, dyslipidemia, polyuria, reduced body weight gain, polydipsia and polyphagia [2]. Contractile dysfunction, which is frequently observed in the diabetic heart, may include disturbances in heart rate, stroke volume, cardiac output, ejection fraction and rates of pressure development and relaxation rate [3-7]Experiments performed in ventricular myocytes isolated from diabetic heart have variously displayed either unaltered or reduced amplitude of shortening, and prolonged time course of contraction and relaxation [8-11]. Intracellular free Ca2+ [Ca2+]i transport systems are essential to the initiation and regulation of cardiac myocyte contraction and relaxation. During the process of ECC, the arrival of an action potential at a ventricular myocyte causes membrane depolarization leading to the opening of voltage-gated L-type Ca2+ channels and a small influx of Ca2+. This small entry of Ca2+ triggers a much larger release of Ca2+ from the sarcoplasmic reticulum (SR). There follows a transient rise in intracellular Ca2+ (Ca2+ transient) which binds to the troponin C and initiates and regulates the process of myocyte contraction. Relaxation of contraction begins when Ca2+ is pumped back into the SR via the SR Ca2+ ATPase pump (SERCA2) and effluxed from the cell, primarily via the Na+ /Ca2+ exchanger [12-14]. Experiments performed in cardiac myocytes from diabetic heart have variously displayed disturbances in Ca2+ transient, time course of Ca2+ transient, L-type Ca2+ current, SR Ca2+ content, uptake and release and Na+ /Ca2+ exchange current[5,15-18]. The aim of this review was to evaluate our current understanding ofcontractile dysfunction and disturbances in Ca2+ transport in the STZ-induced diabetic rat heart.

Diabetogenic Action of Streptozotocin

The general chemical structure of STZ is shown in Figure 1

Figure 1 Diagram showing the chemical structure of streptozotocin

 STZ is an antimicrobial agent which has also been used as a chemotherapeutic alkylating agent [19,20]. STZ is typically administered dissolved in a citrate buffer by either intraperitoneal or intravenousinjection. Once in the blood circulation, STZ travels to the pancreas where it selectively accumulates in pancreatic beta cells via the low-affinity GLUT2 glucose transporter in the plasma membrane. It should be noted that STZ is also able to damage other organs that express GLUT2 including the kidney and the liver [21,22]. It is generally believed that the toxicity of STZ is dependent upon the DNA alkylating activity of its methylnitrosourea moiety and methylation of the DNA molecule. Transfer of the methyl group from STZ to the DNA molecule causes damage and fragmentation of the DNA. Beta cell toxicity might also arise from the action of reactive oxygen species [1,23]. Whatever the mechanism(s) of action, the consequences of STZ treatment are damage to the beta islet cells of the pancreas and a reduction in the ability of these cells to produce insulin (Figure 2).

Figure 2 A schematic diagram showing the toxic effects of streptozotocin in beta cells. Adapted from Lenzen, 2008(1)

The nature of the pathophysiological effects of STZ depends to a large extent on the dose and duration of treatment.

Blood Chemistry in the Streptozotocin-Induced Diabetic Rat

The general characteristics of the STZ-induced diabetic rat include hypoinsulinemia, hyperglycemia, polyuria, decreased body weight gain, polydipsia and polyphagia [2]. Insulin levels fall dramatically in diabetic rats compared to controls (Table 1).

Due to the falling concentrations of insulin, target organs like the liver and muscle are unable to clear glucose from the blood circulation and blood glucose rises. Following a 45-60 mg/kg body weight injection of STZ in adult rats the blood glucose levels typically rise to an average of 400 mg/dl in STZ treated rats compared to 100 mg/dl in controls (Table 2).     

Urine glucose is also elevated in diabetic rats [24] compared to controls. Persistently elevated levels of glucose in the blood of diabetic rats causes glycosylation of hemoglobin and glycosylated hemoglobin is a widely used diagnostic criterion for DM (Table 3).

Glycosylated hemoglobin can rise to an average of 8.5% in diabetic rats compared to 4.0 % in controls (Table 3). Alterations in lipid profile including increased cholesterol [7], increased free fatty acids [7], increased atrial and brain natriuretic peptides[10,25], reduced insulin-like growth factor-1 and triiodothyronine [7,26], increased creatine[27], and increased blood osmolarity also occur in diabetic animals compared to controls [28,29].

Body weight gain is typically reduced in diabetic rats. 

compared to controls (Table 4).

Heart weight is typically reducedbut may occasionally be unaltered in diabetic rats (Table 5).

Heart weight to body weight ratio is generally increased indicating a sign of hypertrophy but may also be unaltered in diabetic rats compared to controls (Table 6).

Other studies have reported unaltered heart weight to tibial length [30], reduced left ventricle weight [31,32] and increased left ventricle to body weight ratio [33] in diabetic rats compared to controls. These data provide evidence of cardiac hypertrophy in the STZ-induced diabetic ra

Hemodynamic Function In vivo and in the Isolated Perfused Heart of the Streptozotocin-Induced Diabetic Rat

Measures of hemodynamic function are shown in Table 7

. Echocardiographic and other techniques have generally reported reduced heart rate [3-6,32,34,35,35-38] but sometimes unaltered heart rate [7,39] in diabetic rats compared to controls (Table 7). Associated with the changes in heart rate electrophysiological measurements of QRS, QT, PQ and PR intervals are generally prolonged [4,5,35,40] and sometimes unaltered [3,35,40] in diabetic rats compared to controls. Cardiac output may either be reduced or unaltered [4-6], stroke volume may be reduced or unaltered [4-6] and ejection fraction was generally reduced [5,6,32,37-39] but may also be unaltered [41] in diabetic rats compared to controls. Percentage fractional shortening was reduced [4-6,32,37-39] in diabetic rat. Left ventricular systolic pressure was reduced [33,35,36,39,42] and left ventricular diastolic pressure was increased [33,35-37,39,42] in diabetic rats compared to controls. Rates of pressure development and decline were lower [5,31,33,35-37,39] in diabetic rat. In the isolated perfused heart the spontaneous heart rate was lower [9,43], rates of pressure development and decline were lower [9] and the time to peak (TPK) and half relaxation (THALF) of pressure were prolonged [9] in diabetic rat heart. Collectively, these in vivo and in vitro experiments provide evidence of disturbances in hemodynamic function including heart rate, left ventricularsystolic and diastolic pressure, rate of pressure development and decline, and cardiac output in the STZ-induced diabetic rat heart compared to controls

Contraction in Ventricular Myocytes from Streptozotocin-Induced Diabetic Rat

The characteristics of ventricular myocyte contraction are shown in Table 8.  

Generally, ventricular myocytes are isolated from heart using a combination of enzymatic and mechanical dispersal techniques [9,16]. The viability, calculated as the percentage of rod shaped compared to round shaped myocytes, was generally lower [4,16,29,44] in myocytes isolated from diabetic rats compared to controls. Resting cell length was generally unaltered [4,5,10,11,28,43,45-49], but sometimes reduced [27] in ventricular myocytes from diabetic rats compared to controls. Cell width was either unaltered [28] or reduced [27], cell thickness was unaltered [27], surface area was increased [10] and calculated cell volume may be unaltered [28] or reduced [27] in myocytes from diabetic rats compared to controls. A recent study reported unaltered length in epicardial (EPI) and endocardial (ENDO) ventricular myocytes from diabetic rat [11]. The time course of shortening was prolonged [4,5,8-11,26,28,29,43,45-51] and the time course for half relaxation was either prolonged [4,5,8,9,11,26,43,45,46,50- 52] or unaltered [10,28,29,47-49] in myocytes from diabetic rats compared to controls. A recent study reported prolonged TPK shortening in EPI and ENDO myocytes and prolonged THALF relaxation of shortening in ENDO but not in EPI left ventricular myocytes from STZ-induced diabetic rat [11]. Rate of development of shortening may be reduced [4,5,9,26,37] or unaltered [45,52] and the rate of decline of shortening may also be reduced [4,5,9,26,37] or unaltered [45,52] in myocytes from diabetic rats compared to controls. Amplitude of shortening may be reduced [4,5,9,26,27,37] or unaltered [8,10,11,28,29,43,45-49] in myocytes from diabetic rat. Increasing frequency of stimulation produces a negative staircase effect that was unaltered in myocytes from STZ-induced diabetic rat compared to control [26,45]. Spontaneous contractions (arrhythmic contractions) were increased in myocytes from diabetic rat [2]. Experiments in sarcomeres have reported reduced fractional shortening, decreased maximal rate of shortening and re-lengthening in diabetic rats compared tocontrols [31]. Collectively, these experiments provide evidence of disturbances in the time course of shortening, relaxation of shortening, and amplitude of shortening in ventricular myocytes from STZ-induced diabetic rats compared to age-matched healthy controls.

Excitation-Contraction Coupling in Ventricular Myocytes

The arrival of an action potential at a ventricular myocyte causes depolarization of the myocyte plasma membrane and opening of L-type Ca2+ channels. A small entry of Ca2+ through the Ca2+ channels triggers a large release of Ca2+ from the SR via the SR Ca2+ release channels (Ryanodine receptors). There is a transient rise in intracellular Ca2+ concentration, which binds to troponin C and triggers and regulates the process of myocyte contraction. The process of myocyte relaxation takes place when Ca2+ is pumped back into the SR via the SERCA pump and extruded from the myocyte, primarily via the Na+ /Ca2+ exchanger, and also to lesser extents via the plasma membrane Ca2+ ATPase ] [12-14]. Alterations in intracellular [Ca2+]i and the mechanisms that control intracellular [Ca2+]i including L-type Ca2+ current, SR

Ca2+ transport and Na+ /Ca2+ exchange might partly underlie the disturbances reported in myocyte shortening.

Calcium Transport in Ventricular Myocytes from the Streptozotocin-Induced Diabetic Rat

The effects of STZ-induced diabetes on intracellular Ca2+ are shown in

Table 9.

Resting [Ca2+]i was generally unaltered [8,9,11,18,28,43,44,47-49,51,53] but sometimes reduced [15,26,44,52,52,54] in ventricular myocytes from diabetic rats compared to controls. A recent study reported unaltered resting Ca2+ in EPI and ENDO myocytes in EPI and ENDO left ventricular myocytes from STZ-induced diabetic rats compared to controls [11]. TPK Ca2+ transient was either prolonged [9,17,28,49,55,56] or unaltered [10,16,29,38,43,47,48] in myocytes from diabeticrats. THALF decay of the Ca2+ transient was generally prolonged [4-6,8-10,16,17,26,28,29,38,48-52,54-57] in myocytes from diabetic rats compared to controls. A recent study reported prolonged TPK Ca2+ transient and prolonged THALF decay of the Ca2+ transient in ENDO, but not in EPI left ventricular myocytes from STZ-induced diabetic rat [11]. Amplitude of the Ca2+ transient was either unaltered [11,16,18,28,29,43,46- 49,53,55] or lowered[4-6,9,15,17,26,27,31,32,37,38,54,56,57] in ventricular myocytes from diabetic rats. In addition, the rate of Ca2+transient development and decay were lower in ventricular myocytes from STZ-induced diabetic rats compared to controls [4-6,9,37]. Collectively, these experiments provide evidence of disturbances in the time course of development and decay of the Ca2+transient and the amplitude of the Ca2+ transient inventricular myocytes from STZ-induced diabetic rats compared to controls

Sarcoplasmic Reticulum Calcium in Ventricular Myocytes from the Streptozotocin-Induced Diabetic Rat

The effects of STZ-induced diabetes on SR Ca2+ transport are shown in Table 10.

Rapid application of caffeine stimulates release of Ca2+ from the SR and provides a measure of releasable SR Ca2+ content [58]. Generally, caffeine-evoked Ca2+ transients were reduced [4,5,9,15,17,32,41,44,54,57] or sometimes unaltered [11,16,46] in ventricular myocytes from diabetic rats compared to controls. The rate of rise of the caffeine-evoked Ca2+ transient was slower [4,5,9] in diabetic rat. TPK caffeine-evoked Ca2+ transient was either prolonged [9] or unaltered [16], while the rate of decay of the caffeine-evoked Ca2+ transient was generally decreased [5,9,16,44,46] or sometimes unaltered [30] in diabetic rats compared to controls. In contrast, the THALF decay of the caffeine-evoked Ca2+ transient was prolonged [4,9,16,29] in ventricular myocytes from diabetic rats compared to controls. The recovery rate of electrically-evoked Ca2+ transients following application of caffeine was generally unaltered [11,15,28] or sometimes increased [46]. SR fractional Ca2+ release (electricallyevoked as a fraction of caffeine-evoked Ca2+ transient) was unaltered [16,28,46,48] in ventricular myocytes from diabetic rats compared to controls. Ca2+-stimulated ATPase activity was generally decreased [26,41], but sometimes unaltered [7], SR Ca2+ATPase mRNA was reduced [33] and SR Ca2+ ATPase protein was generally reduced [9,30,32,39,54] or unaltered [5,37] in diabetic rats compared to controls. Ryanodine receptor (RyR) mRNA was unaltered [4]and RyR protein was either reduced [9,17,32,39,54,56] or unaltered [4-6,37] in diabetic rats compared to controls. Moreover, RyR number of functional receptors was decreased [4] RyR activity was decreased [37] and RyR sensitivity to Ca2+ was increased [4,5] in diabetic rats compared to controls. In general, the RyR receptors, which conveyed less current, were more responsive to Ca2+, but instead had reduced threshold for Ca2+ activation and displayed gain of function [6]. In addition calsequestrin mRNA and calsequestrin total protein content were either reduced [33]or unaltered [9,59] in diabetic rats compared to controls

Calcium Sparks in Ventricular Myocytes from the Streptozotocin-Induced Diabetic Rat 

 Calcium sparks represent SR Ca2+ release from clusters of ryanodine receptors. The peak amplitude of sparks was either unaltered [17,57], lower [4,5,41] or increased [39] in myocytes from diabetic rat compared to controls. In addition, the spark frequency was either increased [5,6,17,57], reduced [39,41] or unaltered [4], and the duration of sparks was also unaltered [4,5] or reduced [6] in diabetic rats compared to controls. The TPK amplitude of sparks was either prolonged [17,57] or unaltered [39] and the rate of Ca2+ rise was either shorter [4-6] or unaltered [41] in myocytes from diabetic hearts compared to controls. There was also evidence that the THALF decay of sparks were either prolonged [5,17,39,57], or unaltered [6] and SR Ca2+ load was reduced [39] in ventricular myocytes from diabetic rats compared to controls.

L-type Calcium Current and Sodium/Calcium Exchange Currents in Ventricular myocytes from the Streptozotocin-Induced Diabetic Rat

The effects of STZ-induced diabetes on L-type Ca2+ current and Na+/Ca2+ exchange are shown in Table 11

and 12.

Ventricular myocyte capacitance, which provides a measure of cell size, has been variously reported as either unaltered [17,57,60], increased [18], or decreased [9] in ventricular myocytes from diabetic rats compared to controls. Entry of Ca2+ current via the L-type Ca2+ channels provides the primary trigger for SR Ca2+ release. The density of L-type Ca2+ current across a range of test voltages were either unaltered [2,4,11,57,60,61], or reduced [62,63]in myocytes from diabetic hearts compared to controls. Steadystate inactivation [11,60] and recovery from inactivation [11] are unaltered in ventricular myocytes from diabetic rats compared to controls. Some studies have simultaneously measured L-type Ca2+ current and either shortening or Ca2+ transients. In these studies, L-type Ca2+ current and shortening were reduced [16,29], and L-type Ca2+ current was unaltered and Ca2+ transient was reduced in myocytes from diabetic rats compared to controls[9,41]. Collectively, these experiments provide strong evidence that L-type Ca2+ current may be unaltered or reduced and that changes in L-type Ca2+ current may or may not be associated with changes in shortening or Ca2+ transient in ventricular myocytes from STZ-induced diabetic rat compared to control. Similarly, the Na+ /Ca2+ exchange current density was reduced [18,41,63], and the current decay was prolonged in myocytes from diabetic rats compared to controls [63]. These alterations in amplitude and kinetics of current were associated with reduced [18] Na+ / Ca2+exchange mRNA and generally reducedNa+ /Ca2+ exchange protein [9,18,39,54] in diabetic rat hearts compared to controls.

Myocardial Fibrosis in Ventricular Myocytes from the Streptozotocin-Induced Diabetic Rat

During development of DC, which is caused by hyperglycemia, and the resulting changes in contraction in vivo, the heart responds by producing the transforming growth factor beta 1, which is an initiator and regulator of myocardial fibrosis. Another factor that is also involved in fibrosis is the connective tissue growth factor. Both factors were increased in STZ-induced diabetic hearts compared to controls [36]. Fibrosis was also enhanced [31] with a marked increase in type 1 collagenin diabetic hearts compared to controls [30].

Figure 3

Figure 3 Diagram showing the proposed actions of STZ-induced diabetes on Ca2+ transport systems in the ventricular myocyte. -=No effect, ↓=Reduced activity, ↑=Increased activity

summarizes some of the mechanisms of Ca2+ transport that are altered in STZ-induced diabetic rat heart. It is well known that STZ (45-65 mg/kg ip or iv), administered to adult rats, damages pancreatic beta cells and reduces the capacity of these cells to release insulin resulting in hyperglycemia and various other characteristics that are observed in DM. In vivo, and to a certain extent in the isolated perfused heart, contractile function, in terms of amplitude and kinetics of contraction, are frequently disturbed in the STZ-induced diabetic rat heart. These contractile disturbances are due to many factors including alterations in cellular Ca2+ homeostasis, disorganization of the structureof the myocytes, apoptosis, endothelial and mitochondrial dysfunctions, fibrosis and remodeling of the myocardium. In turn, these endogenous processes lead to hemodynamic dysfunction including reduced cardiac output, reduced stroke volume and ejection fraction, reduced percentage of fractional shortening, reduced systolic and increased diastolic pressure, and lower rate of pressure development and decline. In ventricular myocytes, the amplitude of shortening is frequently reduced and the time course of shortening and re-lengthening may be prolonged (delayed contraction) and this can be partly attributed to derangement in cellular Ca2+ transport. Alterations in cellular Ca2+transport,including disturbances in L-type Ca2+current(the primary trigger for SR Ca2+ release), Na+ /Ca2+ exchange current (the major pathway for Ca2+ efflux from the cell), SR Ca2+ content and SR Ca2+ uptake and release mechanism(s) all contribute to contractile dysfunction in the STZ-induced diabetic rat heart.

 Grants from the College of Medicine & Health Sciences, United Arab Emirates University, Al Ain; United Arab Emirates University, Al Ain; Sheikh Hamdan Bin Rashid Al Maktoum Award, Dubai; Zayed University, Abu Dhabi.

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