Effects of Obesity and Diabetes on Excitation-Contraction Coupling in Zucker Rat Cardiomyocytes

Abstract

Introduction

Diabetes mellitus (DM) is a serious global health problem and obesity is a major risk factor for DM. Cardiovascular complications are a major cause of morbidity and mortality in diabetic patients and electromechanical dysfunction has been widely reported in the diabetic heart. The aim of this study was to investigate the effects of obesity and diabesity on ventricular myocyte shortening and Ca²⁺ signaling in Zucker fatty (ZF) and Zucker diabetic fatty (ZDF) rats, compared to Zucker lean (ZL) rats.

Materials and Methods

Ventricular myocytes were isolated by enzymatic and mechanical dispersal. Myocyte shortening, L-type Ca²⁺ current, and intracellular Ca²⁺ dynamics were investigated with video imaging, whole cell patch clamp, and fluorescence photometry techniques, respectively.

Results

Time to peak (TPK) shortening was prolonged in ZDF (158.59 ± 3.05 ms) compared to ZF (130.33 ± 2.57 ms) and ZL (126.54 ± 3.09 ms) myocytes. The TPK Ca²⁺ transient was prolonged in ZF (67.26 ± 5.69 ms) compared to ZL (51.54 ± 2.32 ms) myocytes and the time to half (THALF) decay of the Ca²⁺ transient was prolonged in ZDF (155.35 ± 2.92 ms) compared to ZF (131.11 ± 3.26 ms) and ZL (129.17 ± 3.12 ms) myocytes. TPK and THALF decay of caffeine-evoked Ca²⁺ transients were prolonged in ZDF compared to ZF and ZL myocytes.

Conclusion

Although the amplitude of shortening was generally well preserved in ZF and ZDF compared to ZL myocytes, the TPK shortening was prolonged in ZDF myocytes, which might partly be explained by defective uptake and release of sarcoplasmic reticulum Ca²⁺ in ventricular myocytes from the ZDF rat.

 

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Keywords

ventricular myocytes - shortening - intracellular Ca²⁺ - L-type Ca²⁺ current - Zucker fatty rat - Zucker diabetic fatty rat - Zucker lean rat

Introduction

Diabetes mellitus (DM) is a serious global health problem and obesity is a major risk factor for DM. In 2021, 537 million adults between the ages of 20 to 79 years had DM globally and this number is expected to increase to 643 million by 2030 and around 783 million by the year 2045.[1] Obesity, which is defined as “abnormal or excessive fat accumulation that may impair health,” has almost tripled since 1975. In 2016, more than 1.9 billion adults aged 18 years and older were overweight and over 650 million were obese globally.[2] Diabetes and obesity are risk factors for cardiovascular disease, which is the main cause of morbidity and mortality in these patients.[3] People who are obese are more likely to develop type 2 DM (T2DM). In T2DM, the body may synthesize and release sufficient insulin into the circulation, however, the cells in the body become resistant to the action of insulin.[4] The term “diabesity” was created due to the strong link between obesity and diabetes. Although most individuals with T2DM are obese, only a small fraction of obese individuals develop T2DM.[5]

In recent years, scientists have developed a novel experimental model of T2DM and obesity called the Zucker diabetic fatty (ZDF) rat.[6] ZDF rats were derived from the Zucker fatty (ZF) (fa/+ ) male rats, which inherit obesity as an autosomal Mendelian recessive trait. The ZF rat has a missense mutation (fatty, fa) in the leptin receptor gene (Lepr), which leads to hyperphagia and the development of obesity without DM. The ZDF homozygous (fa/fa) male rats develop DM as early as 10 weeks of age, reaching 100% incidence by 21 weeks of age.[7] As such, it is an ideal model to understand how obesity-induced T2DM can lead to diabetic cardiomyopathy, which is a major adverse complication of T2DM characterized by defects in both systolic and diastolic function.

Alterations in cardiac Ca²⁺ signaling vary across different experimental models of obesity and diabetes.[8] The aim of this study was to investigate ventricular myocyte shortening and intracellular Ca²⁺ transport in the ZDF and ZF compared to Zucker lean (ZL) control rats.

 

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Materials and Methods

Animals

Experiments were performed in 33 ZDF, 35 ZF, and 36 ZL male rats (Charles River Laboratories, Margate, Kent, United Kingdom) and experiments commenced at 195 days of age. Rats were maintained under a 12-hour light/12-hour dark cycle with free access to water and standard rat diet. A glucose tolerance test was administered before the start of the experiments. After an overnight fast, baseline blood glucose was measured. Blood was collected from the tail vein of nonanesthetized rats. Animals were then injected according to their body weights with glucose (2 g/kg body weight, intraperitoneal) and blood glucose was measured at 30, 60, 120, and 180 minutes after glucose injection. Body weight, heart weight, and nonfasting blood glucose (OneTouch Ultra 2, LifeScan) were measured immediately prior to each cell isolation. Ethical approval for this study was obtained from the Animal Ethics Committee, College of Medicine & Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates.

 

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Isolation of Ventricular Myocytes

Left ventricular myocytes were isolated from the rats according to previously described techniques.[9] In brief, the animals were euthanized using a guillotine and hearts removed rapidly and mounted for retrograde perfusion according to the Langendorff method. After euthanasia, whole blood was collected in ethylenediaminetetraacetic acid containing tubes to prevent blood clotting, centrifuged at 1,200 revolutions per minute (rpm) (C2 series, Centurion Scientific) for 5 minutes, the supernatant blood plasma was removed, and stored at –80°C for insulin enzyme-linked immunosorbent assay (ELISA) measurements. Hearts were perfused at a constant flow of 8 mL/g/heart/min and at 36 to 37°C with cell isolation solution containing in mmol/L: 130 NaCl, 5.4 KCl, 1.4 MgCl₂, 0.75 CaCl₂, 0.4 NaH₂PO₄, 5.0 HEPES, 10 glucose, 20 taurine, and 10 creatine (pH 7.3). Perfusion flow rate was adjusted to allow for differences in heart weight between animals. When the heart had stabilized perfusion was continued for 4 minutes with Ca²⁺-free cell isolation solution containing 0.1 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and then for 6 minutes with cell isolation solution containing 0.05 mmol/L Ca²⁺, 0.75 mg/mL type 1 collagenase (Worthington Biochemical Corp, Lakewood, New Jersey, United States), and 0.075 mg/mL type XIV protease (Sigma, Taufkirchen, Germany). Left ventricle tissue was excised from the heart, minced, and gently shaken for 4 minutes at 36 to 37°C in collagenase-containing isolation solution supplemented with 1% bovine serum albumin. The cell suspension was then filtered using 300 µm nylon mesh (Cadisch Precision Meshes, London, England). The filtrate was centrifuged at 1,000 rpm for 60 seconds. The supernatant was then removed, and the cell pellet was resuspended in cell isolation solution containing 0.75 mmol/L Ca²⁺. This process was repeated four times.

 

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Measurement of Ventricular Myocyte Shortening

Ventricular myocyte shortening was measured according to previously described techniques.[9] In brief, ventricular myocytes were allowed to settle on the glass bottom of a Perspex chamber mounted on the stage of an inverted Axiovert 35 microscope (Zeiss, Göttingen, Germany). Cells were perfused (3–5 mL/min) with normal Tyrode containing the following in mmol/L: 140 NaCl, 5 KCl, 1.0 MgCl₂, 10 glucose, 5 HEPES, and 1.8 CaCl₂ (pH 7.4) at 35 to 36°C. Shortening was measured in electrically stimulated (1 Hz) ventricular myocytes with an IonOptix MyoCam imaging system (IonOptix Corporation, Milton, Massachusetts, United States). Resting cell length (RCL), time to peak (TPK) shortening, time to half (THALF) relaxation, and amplitude (AMP) of shortening were measured. Data were acquired, filtered, and analyzed with IonWizard 6.6 version 10 software (IonOptix LLC, Westwood, Massachusetts, United States).

 

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Measurement of Intracellular Ca²⁺ Concentration

Intracellular Ca²⁺ concentration and sarcoplasmic reticulum (SR) Ca²⁺ were assessed according to previously described techniques.[9] In brief, myocytes were loaded with the fluorescent indicator fura-2 AM (F-1221, Molecular Probes, Eugene, Oregon, United States). Note that 6.25 µL of a 1.0-mmol/L stock solution of fura-2 AM dissolved in dimethyl sulfoxide was added to 2.5 mL of cells to give a final fura-2 concentration of 2.5 µmol/L. Myocytes were shaken gently for 10 minutes at room temperature (22–26°C). After loading with fura-2 AM, myocytes were centrifuged at 1,000 rpm for 60 seconds, washed with normal Tyrode to remove extracellular fura-2 AM, and then left for 30 minutes to ensure complete deesterification of the intracellular fura-2. To measure intracellular Ca²⁺ concentration myocytes were alternately illuminated by 340 and 380 nm light using a fast monochromator (Cairn Research, Faversham, United Kingdom), which 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 fluorescence emitted at the two excitation wavelengths (340/380 ratio) provided an index of intracellular Ca²⁺ concentration. Resting fura-2 ratio, TPK Ca²⁺ transient, THALF decay of the Ca²⁺ transient, and the AMP of Ca²⁺ transients were measured in electrically stimulated (1 Hz) myocytes maintained at 35 to 36°C. Data were acquired and analyzed with IonWizard 6.6 software (IonOptix LLC). After establishing steady-state Ca²⁺ transients in electrically stimulated (1 Hz) myocytes, stimulation was paused for a period of 5 seconds. Caffeine (20 mM) was then applied for 10 seconds using a rapid solution switching device.[10] Electrical stimulation was then resumed and the Ca²⁺ transients were allowed to recover to steady state. Fractional release of SR Ca²⁺ was assessed by comparing the amplitude of the electrically evoked steady-state Ca²⁺ transients with that of the caffeine-evoked Ca²⁺ transient. Ca²⁺ refilling of SR was assessed by measuring the rate of recovery of electrically evoked Ca²⁺ transients following application of caffeine.

 

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Measurement of L-Type Ca²⁺ Current

L-type Ca²⁺ current was measured according to previously described techniques.[11] In brief, L-type Ca²⁺ current was recorded with an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, California, United States). The analog signal was filtered using a four-pole Bessel filter with a bandwidth of 2 kHz and digitized at a sampling rate of 10 kHz under software control (pClamp 10.6.2.2, Molecular Devices). Patch pipettes were fabricated from filamented TW150F-4 4 IN thin-wall borosilicate glass 1.5 OD/1.2 ID (World Precision Instruments, Florida, United States). The cell chamber solution contained the following in mmol/L: 4 NaCl, 110 TEA-Cl, 4 CsCl, 1 MgCl₂, 2 CaCl₂, 10 HEPES, and 10 glucose (pH 7.4 with CsOH). The pipette solution contained the following in mmol/L: 140 CsCl, 2 MgCl₂, 10 TEA-Cl, 10 EGTA, 10 HEPES, and 2 MgATP (pH 7.25 with CsOH). Electrode resistances were 3 to 5 MΩ, and seal resistances were 1 to 5 GΩ. Experiments were performed at room temperature (22–26°C). The current–voltage relationship was obtained by applying 1,000 ms long pulses to voltages between –60 and +50 mV from a holding potential of –50 mV. The steady-state inactivation was measured at a test potential of 0 mV after the 1,000-ms-long prepulses to voltages between –60 and +50 mV. Recovery from inactivation was measured using a two-pulse protocol, in which a 1,000-ms pulse was compared with 50 ms depolarizing pulses from –50 to 0 mV that were separated by interpulse intervals of different duration intervals. The peak Ca²⁺ current amplitude measured during the second pulse was normalized to the current measured by the first pulse and their ratio was plotted against the interpulse interval. Data were acquired and analyzed with pClamp software v 10.6.2.2 (Molecular Devices).

 

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Statistics

Results were expressed as the mean ± standard error of the mean of “n” observations. Statistical comparisons were performed with IBM SPSS and Origin 9.0 (OriginLab, Northampton, Massachusetts, United States) statistics software using either the independent samples t-test or one-way analysis of variance followed by Bonferroni-corrected t-tests for multiple comparisons, as appropriate. p-Values of less than 0.05 were considered to indicate a significant difference.

 

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Results

General Characteristics

The general characteristics of the ZDF, ZF, and ZL rats are shown in [Table 1]. The ZF and ZDF groups exhibited significantly (p = 0.001) increased body weight and heart weight compared to ZL rats. Nonfasting blood glucose was significantly elevated in ZDF (384.44 ± 20.28 mg/dL, p = 0.001) compared to ZF (140.00 ± 4.58 mg/dL) and ZL (118.25 ± 2.62 mg/dL) rats. Nonfasting blood glucose was not significantly (p = 0.731) different between ZF and ZL rats. Heart weight/body weight ratio was significantly increased in ZF (3.27 ± 0.09 g/mg, p = 0.001) compared with ZL (2.71 ± 0.07 g/mg) and ZDF (2.79 ± 0.05 g/mg) rats. Insulin was measured by ELISA in blood plasma using a commercially available rat insulin ELISA kit (10-1250-01, Mercodia rat Insulin ELISA). ZF rats (12.70 ± 1.15 µg/L) had significantly (p = 0.001) higher insulin levels compared to ZL (2.09 ± 0.57 µg/L) and ZDF (2.14 ± 0.29 µg/L) rats and these results are similar to a previously published work.[12]

Table 1 General characteristics of Zucker rats

	ZL (n)	ZF (i)	ZDF (n)
Body weight (g)	439.82 ± 6.46 (33)	695.85 ± 14.30 (33)[a]	558.83 ± 21.44 (36)[a] [b]
Heart weight (g)	1.66 ± 0.05 (33)	2.15 ± 0.05 (33)[a]	2.00 ± 0.06 (36)[a]
Nonfasting blood glucose (mg/dL)	118.25 ± 2.62 (32)	140.00 ± 4.58 (33)	384.44 ± 20.28 (36)[a] [b]
Heart weight/Body weight ratio (mg/g)	2.71 ± 0.07 (33)	3.27 ± 0.09 (33)[a]	2.79 ± 0.05 (36)[b]
Plasma insulin (µg/L)	2.09 ± 0.57 (10)	12.70 ± 1.15 (9)[a]	2.14 ± 0.29 (8)[b]

Abbreviations: SEM, standard error of the mean; ZDF, Zucker diabetic fatty; ZF, Zucker fatty; ZL, Zucker lean.

Note: Data are mean ± SEM, number of animals is in parenthesis (n). Data provided in this table was adapted from Sultan et al.[37]

^a p < 0.05 compared to ZL.

^b p < 0.05 compared to ZF.

 

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Glucose Tolerance Test

The glucose tolerance test was performed just before commencement of experiments and the results are shown in [Fig. 1]. The fasting blood glucose was highest in ZDF (223.4 ± 12.86 mg/dL, n = 35), intermediate in ZF (133.53 ± 2.99 mg/dL, n = 34), and lowest in ZL (94.55 ± 1.59 mg/dL, n = 33) rats and these differences were significant (p = 0.001). At 180 minutes following the glucose challenge, blood glucose remained significantly (p = 0.001–0.009) elevated in ZDF (373.97 ± 23.86 mg/dL, n = 33) and ZF (212.29 ± 14.43 mg/dL, n = 34) compared to ZL (113.88 ± 4.32 mg/dL, n = 33–35) control rats.

Fig. 1 Glucose tolerance test. Baseline fasting blood glucose was highest in Zucker diabetic fatty (ZDF), intermediate in Zucker fatty (ZF), and lowest in Zucker lean (ZL) rats. Blood glucose measured 120 minutes after glucose challenge remained high in ZDF, was intermediate in ZF, and was approaching baseline in ZL rats. Data are mean ± standard error of the mean (SEM), n = 33 ZL, 34 ZF, and 35 ZDF rats. Horizontal lines above bars represent significant differences. Numbers in parenthesis represent significant differences. Data provided in this figure was adapted from Sultan et al.[37]

 

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Ventricular Myocyte Shortening

The mean RCL was significantly (p < 0.007) longer in ZDF (117.93 ± 0.99 µm) compared to ZL (112.58 ± 1.42 µm) myocytes ([Fig. 2B]). The mean TPK shortening was significantly (p < 0.001) prolonged in ZDF (158.59 ± 3.05 ms) compared to ZF (130.33 ± 2.57 ms) and ZL myocytes (126.54 ± 3.09 ms) ([Fig. 2C]). THALF relaxation of shortening ([Fig. 2D]) and AMP shortening ([Fig. 2E]) were not significantly (p > 0.05) altered in ZDF or ZF compared to ZL myocytes. Intracellular Ca²⁺ signaling is integral to the process of myocyte contraction. Further experiments were performed to investigate if changes in intracellular Ca²⁺ might partly underlie the changes in myocyte shortening.

Fig. 2 Electrically evoked ventricular myocyte shortening. Time to peak (TPK) was significantly prolonged in Zucker diabetic fatty (ZDF) compared to Zucker fatty (ZF) and Zucker lean (ZL) myocytes. Typical records of ventricular myocyte shortening in an electrically stimulated cell (A), resting cell length (B), TPK shortening (C), time to half (THALF) relaxation of shortening (D), and amplitude (AMP) of shortening (E). Data are mean ± standard error of the mean (SEM). n = 79 ZL, 92 ZF, and 87 ZDF cells from 13 ZL, 16 ZF, and 16 ZDF hearts. Horizontal lines above bars represent significant differences.

 

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Ventricular Myocyte Intracellular Ca²⁺

The experimental protocol to investigate Ca²⁺ transients and SR Ca²⁺ is shown in [Fig. 3A]. The mean resting fura-2 ratio (340/380 nm) was significantly (p = 0.022) increased in ZF (0.47 ± 0.007 RU) compared to ZL (0.44 ± 0.006 RU) myocytes ([Fig. 3B]). The mean TPK Ca²⁺ transient was significantly (p = 0.020) prolonged in ZF (67.26 ± 5.69 ms) compared to ZL (51.54 ± 2.32 ms) myocytes ([Fig. 3C]). THALF decay of the Ca²⁺ transient was significantly (p < 0.001) prolonged in ZDF (155.35 ± 2.92 ms) compared to ZF (131.11 ± 3.26 ms) and ZL (129.17 ± 3.12 ms) myocytes ([Fig. 3D]). AMP of the Ca²⁺ transient was significantly (p < 0.007) increased in ZF (0.118 ± 0.007 RU) compared to ZDF (0.094 ± 0.004 RU) myocytes ([Fig. 3E]).

Fig. 3 Electrically evoked Ca²⁺ transients and caffeine-evoked Ca²⁺ transients. Time to half (THALF) decay was significantly prolonged in electrically stimulated Zucker diabetic fatty (ZDF) myocytes, time to peak (TPK) and THALF decay of caffeine-evoked Ca²⁺ transients was prolonged in ZDF compared to Zucker fatty (ZF) and Zucker lean (ZL) myocytes. Fractional Ca²⁺ release and recovery of Ca²⁺ transient was reduced in ZDF compared to ZF myocytes. Typical recording to illustrate the experimental protocol in a ventricular myocyte from a ZL rat (A), ES, electrically stimulated; Caff, caffeine stimulated. Resting fura-2 ratio (B), TPK (C), THALF decay (D), and amplitude (AMP) (E) of electrically evoked Ca²⁺ transients. TPK (F), THALF decay (G), and AMP (H) of caffeine-evoked Ca²⁺ transients. Fractional release (I) and recovery of the Ca²⁺ transient (J). Data are mean ± standard error of the mean (SEM). n = 72 ZL, 70 ZF, and 82 ZDF cells from 13 ZL, 16 ZF, and 16 ZDF hearts. Horizontal lines above bars represent significant differences.

TPK of caffeine-evoked Ca²⁺ transients was significantly (p = 0.038) prolonged in ZDF (575.46 ± 30.96 ms) compared to ZF (468.81 ± 29.73 ms) and significantly (p = 0.001) prolonged in ZDF compared to ZL myocytes (377.82 ± 29.41 ms) ([Fig. 3F]). THALF decay of the caffeine-evoked Ca²⁺ transient was also significantly (p = 0.001) prolonged in ZDF (2050.71 ± 76.1 ms) compared to ZF (1638.03 ± 75.2 ms) and in ZDF compared to ZL myocytes (1459.4 ± 60.6 ms) ([Fig. 3G]). AMP of the caffeine-evoked Ca²⁺ transient was not significantly (p > 0.05) altered in ZDF or ZF compared to ZL myocytes ([Fig. 3H]). Fractional release of Ca²⁺ ([Fig. 3I]) was significantly lower (p = 0.002) and recovery of the Ca²⁺ transients was significantly (p = 0.013) lower in the ZDF compared to ZF myocytes ([Fig. 3J]). To further investigate Ca²⁺ signaling, L-type Ca²⁺ current was assessed.

 

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L-Type Ca²⁺ Current

During the process of excitation-contraction coupling (ECC), the arrival of an action potential depolarizes the cardiac myocyte membrane, leading to the opening of L-type Ca²⁺ channels. There is a small entry of Ca²⁺ via the L-type Ca²⁺ channels, which triggers a large release of Ca²⁺ from the SR. The rise of intracellular Ca²⁺ initiates and regulates the process of contraction.[13] Typical records (left panel) and voltage protocols (right panel) of activation (top panel) and inactivation (bottom panel) of L-type Ca²⁺ current in a ventricular myocyte from a ZL rat are shown in [Fig. 4A]. The current/voltage relationship (I-V curve) is shown in [Fig. 4B]. There were no significant (p > 0.05) differences in the amplitude of L-type Ca²⁺ current at test potentials in the range –60 to +50 mV. Steady-state activation of the L-type Ca²⁺ current is shown in [Fig. 4C]. Typical records of L-type Ca²⁺ current in a ventricular myocyte from a ZL rat are shown in [Fig. 4D] (upper panel) and voltage protocol in [Fig. 4D] (lower panel) to investigate recovery from inactivation. Recovery from inactivation at various interpulse intervals with variable duration is shown in [Fig. 4E]. The results indicate that the activation and inactivation were not significantly (p > 0.05) altered in ZDF and ZF compared to ZL myocytes. The mean membrane capacitance was not significantly (p > 0.05) different between ZL (279.53 ± 19.49 pF), ZF (291.24 ± 25.46 pF), and ZDF (263.29 ± 13.45 pF) myocytes.

Fig. 4 L-type Ca²⁺ current. L-type Ca²⁺ current activation, inactivation, and recovery from inactivation were not significantly altered in Zucker diabetic fatty (ZDF) and Zucker fatty (ZF) compared to Zucker lean (ZL) myocytes. Typical records (left panel) and voltage protocols (right panel) of activation (top panel) and inactivation (bottom panel) of L-type Ca²⁺ current in a ventricular myocyte from a ZL rat (A). Current–voltage relationship (B). Steady-state activation of the L-type Ca²⁺ current (C). Data are mean, n = 17 ZL, 17 ZF, and 14 ZDF cells from 3 ZL, 3 ZF, and 3 ZDF rats. Typical records of L-type Ca²⁺ current in a ventricular myocyte from a ZL rat (D, upper panel) and voltage protocol (D, lower panel) to investigate recovery from inactivation. Recovery from inactivation at various interpulse intervals with variable duration (E). Data are mean, n = 16 ZL, 17 ZF, and 13 ZDF cells from 3 ZL, 3 ZF, and 3 ZDF hearts.

 

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Discussion

ZDF and ZF rats were characterized by increased body weight and heart weight compared to ZL rats and the ZDF rats had elevated nonfasting blood glucose compared to ZF and ZL rats. Heart weight/body weight ratio was highest in ZF compared to ZL and ZDF rats. Fasting blood glucose was highest in ZDF, intermediate in ZF, and lowest in ZL rats. At 180 minutes, following the glucose challenge, blood glucose was still significantly elevated in ZDF and in ZF compared to ZL rats. It was interesting to find that blood insulin was significantly raised in ZF rats but not in ZDF rats compared to ZL rats. A previous study also reported raised insulin in ZF rats compared to ZDF and ZL rats.[12] [14] The ZF rat is an obesity/hyperinsulinemic model whereas the ZDF rat is an obesity/hyperglycemic model. The degree of obesity in the ZF rat is substantially greater (58%) than that in the ZDF (27%) rat. The anabolic effects of the raised levels of insulin might contribute to the larger weight gain in ZF rats.

It has been shown that incubation of cardiac myocytes in a culture medium containing high glucose impairs cellular mechanisms contributing to myocardial relaxation and that this impairment may involve glycosylation of nascent proteins.[15] These results might be explained by considering the mechanisms involved in insulin signaling and degradation. The ratio of the bound and unbound insulin to its receptors could be compromised in ZF rat. Insulin signaling starts with binding to a tyrosine kinase receptor called insulin receptor kinase (IRK). Following autophosphorylation, it is internalized into the endosomal system where it recruits glucose transporters from intracellular stores in skeletal, cardiac muscle, and adipose tissue, which in turn activates various biological processes. Consequently, degradation of insulin is controlled by intraendosomal acidification, in which “insulinase” promotes the dissociation of insulin from the IRK. This degradation may also be compromised in the ZF rat.[16] [17]

RCL was longer in ZDF compared to ZF and ZL myocytes and TPK shortening was prolonged in ZDF compared to ZF and ZL myocytes. The increased RCL in ZDF myocytes may indicate hypertrophy in ZDF rat hearts. The force of contraction may be varied by either increasing the amount of free intracellular Ca²⁺, and/or by altering the sensitivity of the myofilaments to Ca²⁺. Increasing the cell length of the muscle also increases the sensitivity of troponin C (a cardiac regulatory protein) to Ca²⁺, which is called “length-dependent Ca²⁺ sensitivity,” and it can also lead to enhanced intracellular Ca²⁺.[18] [19] Evidence from the current study suggests that ZDF is not able to contract as efficiently as ZL and ZF, perhaps due to reduced force of contraction and/or ventricular dilatation. TPK shortening was prolonged in ZDF compared to ZF and ZL myocytes, which might partly be attributed to the increased length of ZDF myocytes. It might also be attributed to alterations in Ca²⁺ transport as well as the velocity of contraction and relaxation. Neither THALF relaxation nor AMP shortening was significantly altered in ZDF or ZF compared to ZL myocytes. Preserved THALF relaxation and AMP of shortening has been previously reported in ZDF myocytes compared to ZL myocytes from rats aged 30 to 34 weeks.[20] In another study, AMP of shortening at 6 and 22 weeks was unchanged and the time course of shortening at 6 weeks was also unaffected. However, the TPK shortening and THALF relaxation of shortening were prolonged in ZDF rats at 22 weeks.[21]

Changes in intracellular Ca²⁺ signaling might partly underlie the prolonged time course of shortening observed in ZDF myocytes. THALF decay of the electrically evoked Ca²⁺ transient was prolonged in ZDF compared to ZF and ZL myocytes. Further investigation of the molecular structure, specifically the SR and the sarcomeres, which are important in ECC, might clarify the cause of the elevated fura-2 ratio in ZF myocytes.[13] [22] [23] [24] [25] The SR consists of terminal cisternae, which are large regions at close proximity to the ends of the transverse tubules known as “T-tubules,” that release Ca²⁺ ions and the longitudinal tubules, which sequester and concentrate Ca²⁺. Evidence suggests that loss of T-tubule integrity can profoundly affect ECC in myocytes, which is evident during action potential propagation.[26] [27] It is important to note that T-tubule density can be measured using whole-cell capacitance in voltage-clamped myocytes relative to cell area, previous results indicate that rat myocytes can be kept in quiescent culture for 24 hours with no detectable detubulation or loss of T-tubules.[26] TPK Ca²⁺ transient was not altered in ZDF compared to ZL myocytes. However, TPK Ca²⁺ transient was prolonged in ZF compared to ZL myocytes. THALF decay of the Ca²⁺ transient was prolonged in ZDF compared to ZF and ZL myocytes. AMP of the Ca²⁺ transient was increased in ZF compared to ZDF myocytes. These disturbances in time course of the Ca²⁺ transient might be partly attributed to defective SR Ca²⁺ transport.

TPK and THALF decay of the caffeine-evoked Ca²⁺ transients were prolonged in ZDF compared to ZF and ZL myocytes as well as significantly lower fractional release of Ca²⁺ and recovery of Ca²⁺ transients in the ZDF compared to ZF myocytes. Collectively, these findings suggest delayed release and uptake of Ca²⁺ by the SR and this in turn might be caused by defective SR Ca²⁺ release channel ryanodine receptor and/or SR Ca²⁺ ATPase (SERCA) pump activity.[28] [29] [30] Previous studies in different experimental models of diabetes have variously reported enhanced diastolic SR Ca²⁺ leakage, lower caffeine-evoked Ca²⁺ release, and decreased rate of SR Ca²⁺-ATPase (SERCA)-mediated Ca²⁺ uptake in myocytes from db/db diabetic mice, as well as lowered AMP of caffeine-releasable Ca²⁺, SR store and rates of Ca²⁺ release, and depressed resequestration into SR in myocytes from streptozotocin (STZ)-induced diabetic rats.[31] [32] Reduced release of Ca²⁺ may be linked to structural defects in the SR Ca²⁺ release channel, and reduced levels of expression of messenger ribonucleic acid (mRNA) and protein for the Ca²⁺ release channel have been reported in type 2 diabetic patients, db/db mice, and STZ- and alloxan-induced diabetic rats.[33] [34] [35] [36]

It is interesting to compare the shortening and intracellular Ca²⁺ data in this study and our previous study in ZDF and ZF myocytes.[37] In this study, myocyte shortening and Ca²⁺ transients were recorded separately. In the Sultan et al study shortening and Ca²⁺ transient were recorded in fura-2-loaded myocytes, simultaneously. Fura-2 loading of the myocytes might have buffered intracellular Ca²⁺, which in turn might account for differences in the time course and amplitude of shortening between the two studies.

Activation, inactivation, and recovery from inactivation were not significantly altered in ZDF and ZF compared to ZL myocytes. The L-type Ca²⁺ results are surprising considering the significant changes in Ca²⁺ signaling. Under these circumstances, changes in current densities as well as changes in activation and inactivation current might be expected. Myocyte contraction is regulated by influx of Ca²⁺ via the L-type Ca²⁺ channels, which provides the trigger for SR Ca²⁺ release. Disturbances of L-type Ca²⁺ current would have implications for the release of SR Ca²⁺. L-type Ca²⁺ current was not significantly altered in ZDF or ZF compared to ZL myocytes over a wide range of test voltages. However, previous studies performed in Zucker rats aged 9 to 13 and 30 to 34 weeks have demonstrated reduction in L-type Ca²⁺ current over a range of test potentials in ZDF myocyte.[20] [38] Another study in ZF rats aged 16 to 17 weeks demonstrated a reduction in L-type Ca²⁺ current and defective Ca²⁺ inactivation.[39] L-type Ca²⁺ current activation, inactivation, and recovery from inactivation were not significantly altered in myocytes from epicardial and endocardial regions in STZ-treated rats or Wistar controls,[40] which was also evident in the current Zucker study. Unaltered L-type Ca²⁺ current and also reduced L-type Ca²⁺ current has also been reported in ventricular myocytes from other experimental models of diabetes including the Goto-Kakizaki rat and the STZ-induced diabetic rat.[41] [42] [43] Differences in results might partly be attributed to differences in animal model, duration of diabetes, and experimental methodology.

Recovery of intracellular Ca²⁺ to resting levels is mainly driven by uptake of Ca²⁺ into the SR and extrusion of Ca²⁺ from the cell via the Na⁺/Ca²⁺ exchange (NCX), operating in forward mode. Defects in NCX might contribute to alterations in the decay of the Ca²⁺ transient.[8] [44] [45] [46] In our study, TPK and THALF decay of the caffeine-evoked Ca²⁺ transients were prolonged in ZDF suggesting there may be defects in SR Ca²⁺ or NCX transport in ZDF myocytes. These results are consistent with a previous study by Lima-Leopoldo et al that showed no upregulation of NCX genes in high-fat diet (obese) Wistar rats.[47] However, Hattori et al showed that STZ-induced diabetic rats had diminished NCX current density compared to control as well as NCX expression and function due to reduced NCX protein and mRNA.[45] STZ can induce near to complete destruction of pancreatic β-cells, very low blood insulin, and very high blood glucose and characteristics of type-1 diabetes, which might not be the case in ZDF rats.

This study has several limitations. These include the potential influence of age selection on outcomes, the effect of myocyte stimulation rate (set at 1 Hz in this study) on results, and the impact of perfusate composition on the function of metabolically distinct myocytes. Additionally, the experiments assessing myocyte shortening and Ca²⁺ transients were conducted at 35 to 36°C, whereas Ca²⁺ current measurements were performed at room temperature, which may introduce variability. Furthermore, to draw definitive conclusions, the weight gain between ZF and ZDF rats should ideally be comparable.

Investigating the roles of other ion transporters including the Na⁺/Ca²⁺ exchanger and the use of pharmacological interventions to modulate SR Ca²⁺ uptake/release could be used to further investigate Ca²⁺ signaling in the diabetic heart.

 

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Conclusion

Amplitude of shortening is generally well preserved in ZDF myocytes. Defects in the uptake and release of SR Ca²⁺ might partly underlie the altered time course of the Ca²⁺ transient and shortening in ventricular myocytes from ZDF rat. Disturbances in the time course of shortening and Ca²⁺ transient may have implications for the hemodynamic function of the heart. Further molecular, functional, and structural studies will be required to investigate the Ca²⁺ uptake and release mechanisms of the SR.

 

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Conflict of Interest

None.

Acknowledgment

Authors would like to acknowledge Mr. Ammar Mahgoub for his help in taking care of the animals.

Authors' Contributions

F.H. secured funding and made substantial contributions to conception and design, drafting, and revising the article. A.S. and M.Q. made substantial contributions to acquisition of data and analysis of data. A.S. and A.S. made substantial contributions to interpretation and graphical representation of data. All authors contributed to the writing and revision of the manuscript. All authors approve the final version to be published.

Compliance with Ethical Principles

Ethical approval for this study was obtained from the Animal Ethics Committee, College of Medicine & Health Sciences, United Arab Emirates University.

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Publication History

Article published online:
19 May 2025