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Gaber, Jayaprakash, Qureshi, Oz, and Howarth: Well-preserved ventricular myocyte shortening in Goto–Kakizaki type 2 diabetic rats

Introduction

The global prevalence of type 2 diabetes mellitus (T2DM) is projected to rise from 171 million in 2000 to 366 million in 2030.1 In the United Arab Emirates (UAE), a recent study in 2007 reported age-standardized rates for diabetes mellitus and pre-diabetes, among 30- to 64-year-old Emiratis, of 29% and 24%, respectively.2 Risk factors for diabetes mellitus include obesity, sedentary lifestyle, unhealthy eating habits, family history of diabetes mellitus, genetics, increasing age, high blood pressure and high cholesterol. A variety of diastolic and systolic dysfunctions including reduced left ventricular ejection fraction, impaired myocardial velocity at early diastole, abnormal relaxation during the early filling phase, prolonged isovolumetric relaxation, lower peak systolic and early diastolic velocity, impaired diastolic relaxation and filling, and reduced peak filling rate have been reported in hearts of type 2 diabetic patients and the severity of the abnormalities is partially dependent on the age of the patient and the length of time that they have had diabetes mellitus.3 The Goto–Kakizaki (GK) rat, one of the best-characterized genetic animal models of T2DM, was created by selective breeding of an outbred colony of Wistar rats, with selection for high glucose levels in an oral glucose tolerance test.4 T2DM in the GK rat appears to be polygenic with at least three different loci involved in the disease5 and the general characteristics of the GK rat include fasting hyperglycaemia, impaired insulin secretion and insulin resistance.4 In the GK rat there are a variety of cardiac dysfunctions including decreased heart rate, decreased ejection fraction (mainly as a result of a loss in left ventricular longitudinal contraction) and prolonged time of shortening and/or relaxation in ventricular myocytes and some of these cardiac dysfunctions may be attributed to defects in Ca2+ transport.69 Poor diet is a risk factor for many diseases including T2DM, and widespread consumption of sugar-sweetened beverages has been linked to higher incidence of obesity.10,11 Many popular fizzy drinks and other beverages contain large quantities of sucrose,10 and a number of studies have demonstrated that consumption of sucrose-enriched diets can cause insulin resistance, hyperlipidaemia and increased fat deposition in the heart.12,13 Ventricular myocyte shortening and Ca2+ transport in GK type 2 diabetic rats that were fed a sucrose-enriched diet has been investigated.

Methods

Experimental protocol

Male GK and Wistar control rats aged 7 weeks were divided into two subgroups. All animals received normal rat chow and drinking water ad libitum. One subgroup of GK and control rats received normal drinking water for the entire duration of the experiment while the other subgroup received water containing 100 mmol/l sucrose for 2 months, 200 and 300 mmol/l sucrose for 1 month at each concentration and 400 mmol/l sucrose thereafter. Body weight and non-fasting blood glucose were measured immediately prior to experiments and ventricular myocyte experiments began after 6 months of dietary intervention. Approval for this project was obtained from the Animal Ethics Committee, College of Medicine and Health Sciences, UAE University.

Ventricular myocyte isolation

Ventricular myocytes were isolated from GK and control rats according to previously described techniques.14 The animals were euthanized using a guillotine and their hearts removed rapidly and mounted on a Langendorff perfusion system. The hearts were perfused at a constant flow of 8 ml/g per heart per minute at 36–37°C with a cell isolation solution containing 130.0 mmol/l NaCl, 5.4 mmol/l KCl, 1.4 mmol/l MgCl2, 0.75 mmol/l CaCl2, 0.4 mmol/l NaH2PO4, 5.0 mmol/l HEPES, 10.0 mmol/l glucose, 20.0 mmol/l taurine and 10.0 mmol/l creatine (pH 7.3). The perfusion flow rate was adjusted to allow for differences in heart weight between animals. After heart contractions had stabilized, perfusion was continued for 4 minutes with a Ca2+-free cell isolation solution containing 0.1 mmol/l EDTA and then for 6 minutes with cell isolation solution containing 0.05 mmol/l Ca2+, 0.75 mg/ml type 1 collagenase (Worthington Biochemical Corporation, Lakewood, NJ, USA) and 0.075 mg/ml type XIV protease (Sigma, Taufkirchen, Germany). Ventricle tissue was excised from the heart, minced and gently shaken in collagenase-containing isolation solution supplemented with 1% bovine serum albumin. Cells were then filtered from this solution at 4-minute intervals and resuspended in cell isolation solution containing 0.75 mmol/l Ca2+.

Measurement of ventricular myocyte shortening

Ventricular myocyte shortening was measured according to previously described techniques.14 Cells were superfused (3–5 ml/min) with normal Tyrode’s solution containing 140.0 mmol/l NaCl, 5.0 mmol/l KCl, 1.0 mmol/l MgCl2, 10.0 mmol/l glucose, 5.0 mmol/l HEPES and 1.8 mmol/l CaCl2 (pH 7.4). Unloaded myocyte shortening was recorded using a VED-114 video edge detection system (Crystal Biotech, Northborough, MA, USA). Resting cell length, time to peak (TPK) shortening, time to half (THALF) relaxation and amplitude of shortening (expressed as a percentage of resting cell length) were measured in field-stimulated (1 Hz) myocytes maintained at 35–36°C. Data were acquired and analysed with Signal Averager software version 6.37 (Cambridge Electronic Design, Cambridge, UK).

Measurement of intracellular Ca2+ concentration

Myocytes were loaded with the fluorescent indicator fura-2 acetoxymethyl ester (F-1221; Molecular Probes, Eugene, OR, USA) and myocyte shortening was measured according to previously described techniques.14 To measure intracellular Ca2+ concentration, myocytes were alternately illuminated with 340 nm and 380 nm light using a monochromator (Cairn Research, Faversham, UK) that changed the excitation light every 2 ms. The resulting fluorescence emitted at 510 nm was recorded by a photomultiplier tube and the ratio of the emitted fluorescence at the two excitation wavelengths (340/380 ratio) was calculated to provide an index of intracellular Ca2+ concentration. Resting fura-2 ratio, TPK Ca2+ transient, THALF decay of the Ca2+ transient and the amplitude of the Ca2+ transient were measured in field-stimulated (1 Hz) myocytes maintained at 35–36°C. Data were acquired and analysed with Signal Averager software.

Assessment of myofilament sensitivity to Ca2+

In some cells, shortening and fura-2 ratio were recorded simultaneously. Myofilament sensitivity to Ca2+ was assessed from phase-plane diagrams of fura-2 ratio versus cell length by measuring the gradient of the fura-2 cell length trajectory during late relaxation of the twitch contraction.15,16 The position of the trajectory reflects the relative myofilament response to Ca2+ and, hence, can be used as a measure of myofilament sensitivity to Ca2+.

Statistics

Results were expressed as the mean ± standard error of the mean (SEM) of the number of observations. Statistical comparisons were performed using analysis of variance and Bonferroni post hoc test [SPSS (Statistical Product and Service Solutions) version 20; SPSS Inc., Chicago, IL, USA], for which P < 0.05 was considered to indicate a significant difference.

Results

General characteristics of Goto–Kakizaki rats

Experiments were performed after the animals had been receiving a sucrose-enriched diet for a period of 6 months. The general characteristics of the animals are shown in Table 1. Body weight, heart weight and heart weight to body weight ratio were not statistically significantly different among the four groups of animals (P > 0.05). Non-fasting blood glucose was increased in GK compared with the control rats; however, the difference did not reach statistical significance (P > 0.05). Feeding GK rats with sucrose resulted in a statistically significant (P < 0.05) threefold increase in blood glucose compared with control rats. Blood glucose was also significantly elevated in GK/sucrose rats compared with control/sucrose rats (P < 0.05).

TABLE 1

General characteristics of animals

Characteristic Controla Control/sucroseb GK GK/sucrose
Body weight (g) 389.67 ± 22.40 362.33 ± 17.91 425.00 ± 7.09 444.33 ± 18.37
Heart weight (g) 1.30 ± 0.06 1.27 ± 0.03 1.40 ± 0.00 1.47 ± 0.07
Heart weight–body weight (mg/g) 3.35 ± 0.12 3.50 ± 0.10 3.30 ± 0.05 3.30 ± 0.02
Blood glucose (mg/dl) 85.67 ± 3.71 106.67 ± 15.81 157.67 ± 12.81 223.00 ± 30.04

Data are mean ± SEM, n =3  animals.

a P < 0.01 comparing control with GK/sucrose rats.

b P < 0.05 comparing control/sucrose with GK/sucrose rats.

Ventricular myocyte shortening

Figure 1 shows (a) resting cell length, (b) TPK shortening, (c) THALF relaxation of shortening and (d) amplitude of shortening. Resting cell length in myocytes from GK rats (146.84 ± 4.46 µm, n = 27 cells) was significantly longer (P < 0.05) than in myocytes from control/sucrose rats (127.53 ± 4.84 µm, n = 20 cells) (Figure 1a). TPK shortening was not significantly altered (P > 0.05) in myocytes from GK rats compared with control rats or in GK/sucrose rats and control/sucrose rats compared with GK and control rats, respectively (Figure 1b). THALF shortening was not significantly altered (P > 0.05) in myocytes from GK rats compared with control rats or in GK/sucrose and control/sucrose rats compared with GK and control rats, respectively (Figure 1c). Amplitude of shortening was not significantly altered (P > 0.05) in myocytes from GK rats compared with control or in GK/sucrose and control/sucrose compared with GK and control rats, respectively (Figure 1d).

FIGURE 1

Ventricular myocyte shortening in GK and control rats fed sucrose-enriched diets. (a) Resting cell length; (b) TPK shortening; (c) THALF relaxation of shortening; and (d) amplitude of shortening, expressed as a percentage of resting cell length, in myocytes from control, control/sucrose, GK and GK/sucrose rats. Data are mean ± SEM, n = 20–31 cells. Horizontal lines above bars represent significant differences. *P < 0.05.

7-2-5-fig1a.jpg7-2-5-fig1b.jpg7-2-5-fig1c.jpg7-2-5-fig1d.jpg

Ventricular myocyte intracellular Ca2+

Figure 2 shows (a) resting fura-2 ratio, (b) TPK Ca2+ transient, (c) THALF decay of the Ca2+ transient and (d) amplitude of the Ca2+ transient. Resting fura-2 ratio unit (RU) was significantly reduced (P < 0.05) in myocytes from GK/sucrose (0.64 ± 0.02 RU, n = 24 cells) rates compared with myocytes from control rats (0.72 ± 0.02 RU, n = 29 cells) (Figure 2a). TPK Ca2+ transient was significantly (P < 0.05) prolonged in myocytes from GK/sucrose rats (68.68 ± 3.01 ms, n = 22) compared with GK control rats (55.82 ± 2.47 ms, n = 25) and in myocytes from GK/sucrose rats (68.68 ± 3.01 ms, n = 22) compared with myocytes from control/sucrose rats (55.91 ± 2.98 ms, n = 21 cells) (Figure 2b). THALF decay of the Ca2+ transient was not significantly altered (P > 0.05) in myocytes from GK rats compared with control rats or in GK/sucrose and control/sucrose rats compared with GK and control rats, respectively (Figure 2c). There was progressive increase in the amplitude of the Ca2+ transient. The amplitude of Ca2+the transient was smallest in myocytes from control and progressively increased in control/sucrose, GK and was largest in GK/sucrose rats. The amplitude of the Ca2+ transient was significantly larger (P < 0.05) in myocytes from GK/sucrose rats (0.25 ± 0.03 RU, n = 24 cells) than in myocytes from control rats (0.16 ± 0.01 RU, n = 29 cells) (Figure 2d).

FIGURE 2

Ventricular myocyte intracellular Ca2+ in GK and control rats fed sucrose-enriched diets. (a) Resting fura-2 ratio, (b) TPK Ca2+ transient, (c) THALF decay of the Ca2+ transient and (d) amplitude of the Ca2+ transient in myocytes from control, control/sucrose, GK and GK/sucrose rats. Data are mean ± SEM, n = 22–29 cells. Horizontal lines above bars represent significant differences. *P < 0.05.

7-2-5-fig2a.jpg7-2-5-fig2b.jpg7-2-5-fig2c.jpg7-2-5-fig2d.jpg

Myofilament sensitivity to Ca2+

Figure 3a shows a typical record of shortening and Ca2+ transient in a control myocyte. Figure 3b is a phase-plane diagram of fura-2 ratio versus cell length. The gradient of the fura-2 cell length trajectory during late relaxation (500–800 ms) of the twitch contraction was not significantly altered (P > 0.05) in myocytes from GK rats compared with control rats or in GK/sucrose and control/sucrose rats compared with GK and control rats, respectively (Figure 3c).

FIGURE 3

Myofilament sensitivity to Ca2+ in GK and control rats fed sucrose-enriched diets. (a) A typical record of shortening and Ca2+ transient in a control myocyte; (b) a phase-plane diagram of fura-2 ratio vs. cell length; and (c) the gradient of the fura-2 cell length trajectory during late relaxation (500–800 ms) of the twitch contraction. The arrow shows where the gradient was measured. Data are mean ± SEM, n = 17–27 cells.

7-2-5-fig3a.jpg7-2-5-fig3b.jpg7-2-5-fig3c.jpg

Discussion

The experiments investigated ventricular myocyte shortening and Ca2+ transport in GK rats fed a sucrose-enriched diet for a period of 6 months. The major findings were:

  1. Non-fasting blood glucose was increased in GK rats compared with control rats and more so in GK/sucrose rats compared with control rats; however, body weight, heart weight and heart weight to body weight ratio were unaltered by sucrose-enriched diets.

  2. TPK shortening, THALF relaxation of shortening and amplitude of shortening were unaltered in myocytes from GK rats compared with control rats and in myocytes from GK/sucrose and control/sucrose rats compared with GK and control rats, respectively.

  3. TPK Ca2+ transient was prolonged in myocytes from GK/sucrose rats compared with GK rats and in myocytes from GK/sucrose rats compared with control/sucrose rats.

  4. Amplitude of the Ca2+ transient was increased in myocytes from GK/sucrose rats compared with control rats.

  5. Myofilament sensitivity to Ca2+ was unaltered in myocytes from GK rats compared with control rats and in myocytes from GK/sucrose rats and control/sucrose compared with GK and control rats, respectively.

Non-fasting blood glucose was increased in GK rats compared with the control rats and more so in GK/sucrose rats compared with control rats. Previous studies in animals of different ages have reported elevated and unaltered non-fasting blood glucose and unaltered fasting blood glucose in GK rats.68,14 Interestingly, in a study in which GK rats were fed either low-fat/high-carbohydrate or high-fat/low-carbohydrate diets, non-fasting blood glucose was increased in the GK rats that received the high-carbohydrate diet.17 Feeding GK rats a sucrose-enriched diet increased non-fasting blood glucose threefold compared with control rats. Previous studies in which normal healthy rats were fed either starch-enriched or sucrose-enriched diets showed that animals receiving the sucrose-enriched diet had hyperinsulinaemia, hypertriglyceridaemia, mild elevations in plasma glucose and impaired insulin sensitivity (insulin resistance).13,18

Interestingly, ventricular myocyte shortening was well preserved in GK/sucrose rats compared with GK and control rats and in GK rats compared with control rats. Previous studies have demonstrated an increased amplitude of shortening in myocytes from GK rats compared with control rats and occasionally showed unaltered myocyte longitudinal length, reduced shortening, slower TPK twitch and prolonged relaxation in normal healthy rats receiving sucrose-enriched diets.8,13,18,19

The TPK Ca2+ transient was prolonged in myocytes from GK/sucrose rats compared with GK rats and in GK/sucrose rats compared with control/sucrose rats. Previous experiments in normal healthy rats fed a sucrose-enriched diet have demonstrated prolonged decay of the Ca2+ transient, suggesting prolonged cytoplasmic Ca2+ clearing.19 Interestingly, the amplitude of the Ca2+ transient was larger in myocytes from GK/sucrose rats than in control rats. There was a tendency for increasing amplitude of the Ca2+ transient in myocytes in the order control, control/sucrose, GK and, finally, GK/sucrose rats. Previous experiments have demonstrated reduced amplitude of the Ca2+ transient in myocytes from GK rats compared with control rats and an increased amplitude of the Ca2+ transient in myocytes from rats receiving a sucrose-enriched diet.8,13

Measuring the gradient of the fura-2 cell length trajectory during late relaxation of the twitch contraction provides a measure of myofilament sensitivity to Ca2+.16 Myofilament sensitivity was unaltered in myocytes from GK/sucrose rats compared with GK, control/sucrose and control rats. Interestingly, previous experiments performed in our laboratory have demonstrated altered myofilament sensitivity to Ca2+ in myocytes from GK rats compared with controls.8

In conclusion, ventricular myocyte shortening was well preserved despite some alterations in Ca2+ transport in GK rats that received a sucrose-enriched diet for 6 months. Differences in the age of animals and, therefore, the duration of diabetes mellitus may partly explain differences in the characteristics of shortening and Ca2+ transport reported in different studies. Further experiments including a study of L-type Ca2+ current, the primary trigger for sarcoplasmic reticulum Ca2+ release, will be required to clarify the mechanisms underlying alterations in Ca2+ transport.

Acknowledgements

This study was supported by grants from Sheikh Hamdan Awards for Medical Sciences, College of Medicine and Health Sciences, UAE University, and sponsorship from LABCO, partner of Sigma-Aldrich.

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