INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

EVIDENCE FROM BOTH HUMAN and experimental animals has demonstrated the existence of a specific diabetic cardiomyopathy independent of macrovascular coronary artery complications (7, 23). Diabetic cardiomyopathy is believed to contribute to the high incidence of cardiac dysfunction and mortality in both type I and type II diabetes mellitus. Diastolic dysfunction is one of the most prominent mechanical defects associated with diabetic cardiomyopathy and is characterized by decreased compliance and slower rates of myocardial relaxation (19, 20, 23).

Many studies have investigated the characteristics and the mechanisms of diabetic cardiomyopathy with the use of the whole heart, multicellular tissue, or single cardiac cells, by use of chemically (e.g., streptozotocin and alloxan) induced diabetic animal models (6, 7, 9, 13, 18-20, 23, 25). These cell-specific toxins are capable of destroying pancreatic -cells, resulting in permanent diabetes. However, the chemically induced diabetic models are not dependent on exogenous insulin for survival and thus do not entirely resemble type I human diabetes. Also, some investigators have voiced concerns regarding the influence of gender and age on the susceptibility to the toxins in the chemically induced diabetic model (14, 17).

The spontaneously diabetic BioBreed (BB) rat displays a diabetic syndrome consisting of hypoinsulinemia, hyperglycemia, and glycosuria. It is a result of cell-mediated autoimmune destruction of pancreatic -cells, a process that leads to insulin deficiency and subsequently to an increase in blood glucose and plasma free fatty acids. As a model of diabetes that depends on insulin for survival, the BB diabetic rat is the closest counterpart to human type I diabetes. Distinct morphological as well as functional abnormalities have been reported in BB diabetic rat myocardium. These include loss of myofilaments, disruption of mitochondria, dilation of sarcoplasmic reticulum, depressed myocardial contractility, and rate of ventricular relaxation and sarcoplasmic reticulum Ca²⁺ uptake (2, 10, 21, 22) somewhat similar to dysfunctions reported in chemically induced diabetic hearts (7, 19).

This study was designed to determine whether the mechanical abnormalities seen at the whole heart level are attributable to functional abnormalities of isolated ventricular myocytes isolated from BB spontaneously diabetic rats. Myocytes isolated from 1-yr-old spontaneously diabetic prone (BB/DP) rats and their diabetic-resistant littermates (BB/DR) were used in the study.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals. All animal experimentation was conducted in accordance with the humane animal care standards outlined in the National Research Council's Guide for the Care and Use of Laboratory Animals. Male BB/DP and age-matched BB/DR rats at ~60 days of age were obtained from the breeding colony at the University of Massachusetts (Biomedical Research Models). The rats were housed individually in metabolic cages for daily urinary glucose measurements with the use of enzymatic test strips and were allowed free access to standard laboratory rodent chow and tap water. On the onset of type I diabetes, indicated by glucosuria, the BB/DP rats were anesthetized with brevital sodium (50 mg/kg ip; Eli Lilly, Indianapolis, IN), and a sustained-release insulin implant (Linplant, Linshin Canada, Scarbough, ON, Canada) was inserted in the dorsal neck region of each animal. The rats fully recovered from the effects of the anesthetic within 10-15 min. The length of the Linplant was adjusted to maintain chronic moderate hyperglycemia in the BB/DP rats. Blood glucose levels were measured at 0900 three times per week with the use of a glucose monitor (Accu-ChekII, model no. 792; Boehringer Mannheim Diagnostics, Indianapolis, IN). The active life of the Linplant was ~40 days, thereby eliminating the need for daily insulin injections. Reimplantation was performed as described when blood glucose levels exceeded 350 mg/dl. The animals were killed at 1 yr of age. No implantation was conducted at least 7 days before death.

Cell-isolation procedures. Single ventricular myocytes were enzymatically isolated from the hearts by use of the method described previously (19). Briefly, hearts were rapidly removed and perfused (at 37°C) with Krebs-Henseleit bicarbonate (KHB) buffer containing (in mM) 118 NaCl, 4.7 KCl, 1.25 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 10 HEPES, and 11.1 glucose, equilibrated with 5% CO₂-95% O₂. Hearts were subsequently perfused with a nominally Ca²⁺-free KHB buffer for 2-3 min until spontaneous contractions ceased, followed by a 20-min perfusion with Ca²⁺-free KHB containing 223 U/ml collagenase (Worthington Biochemical, Freehold, NJ) and 0.1 mg/ml hyaluronidase (Sigma Chemical, St. Louis, MO). After perfusion, ventricles were removed and minced, under sterile conditions, and incubated with the Ca²⁺-free KHB with collagenase solution for 3-5 min. The cells were further digested with 0.02 mg/ml trypsin (Sigma) before being filtered through a nylon mesh (300 µm) and subsequently separated from the collagenase-trypsin solution by centrifugation (60 g for 30 s). Myocytes were resuspended in a sterile-filtered, Ca²⁺-free Tyrode buffer containing (in mM) 131 NaCl, 4 KCl, 1 MgCl₂, 10 HEPES, and 10 glucose, supplemented with 2% BSA, with a pH of 7.4 at 37°C. Cells were initially washed with Ca²⁺-free Tyrode buffer to remove residual enzyme, and extracellular Ca²⁺ was slowly added back to 1.25 mM. Myocytes with obvious sarcolemmal blebs or spontaneous contractions were not used. Only rod-shaped myocytes with clear edges were selected for recording of mechanical properties or intracellular Ca²⁺ transients as previously described.

Cell shortening/relengthening. Mechanical properties of ventricular myocytes were assessed by use of a video-based edge-detection system (IonOptix, Milton, MA) (19). In brief, cells were placed in a Warner chamber mounted on the stage of an inverted microscope (Olympus, X-70) and superfused (~1 ml/min at 37°C) with a buffer containing (in mM) 131 NaCl, 4 KCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, and 10 HEPES, at pH 7.4. The cells were field stimulated with suprathreshold voltage and at a frequency of 0.5 Hz (3-ms duration) with the use of a pair of platinum wires placed on opposite sides of the chamber connected to a FHC stimulator (Brunswick, NE). The polarity of stimulatory electrodes was reversed frequently to avoid possible build up of the electrolyte by-products. The myocyte being studied was displayed on the computer monitor with the use of an IonOptix MyoCam camera, which rapidly scans the image area at every 8.3 ms such that the amplitude and velocity of shortening/relengthening is recorded with good fidelity. The soft-edge software (IonOptix) was used to capture changes in cell length during shortening and relengthening.

Intracellular fluorescence measurement. A separate cohort of myocytes was loaded with fura 2-AM (0.5 µM) for 15 min, and fluorescence measurements were recorded with a dual-excitation fluorescence photomultiplier system (IonOptix) as previously described (19). Myocytes were placed on an Olympus X-70 inverted microscope equipped with a temperature-controlled (37°C) Warner chamber and imaged through a Fluor ×40 oil objective. Cells were exposed to light emitted by a 75-W lamp and passed through either a 360- or a 380-nm filter (bandwidths were ±15 nm) while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by a photomultiplier tube after cells were first illuminated at 360 nm for 0.5 s and then at 380 nm for the duration of the recording protocol (333-Hz sampling rate). The 360-nm excitation scan was repeated at the end of the protocol, and qualitative changes in intracellular Ca²⁺ concentration were inferred from the ratio of the fluorescence intensity at two wavelengths.

Statistical analyses. For each experimental series, data are presented as means ± SE. Statistical significance (P < 0.05) for each variable was estimated by ANOVA or t-test where appropriate (Systat, Evanston, IL).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

General features of normal and diabetic animals. The 1-yr-old BB/DP rats exhibited significantly lower body weights and higher serum glucose levels compared with the age-matched nondiabetic BB/DR animals. The absolute heart weight and heart-to-body weight ratio were also larger in the diabetic group. Diabetic animals also showed greater proportional liver and kidney weights (Table 1).

Cell shortening and relengthening from BB/DR and BB/DP cardiac myocytes. Sustained spontaneous diabetes did not affect cell length (CL). The average resting CL of ventricular myocytes used in this study was 115 ± 2 and 121 ± 3 µm in BB/DR and BB/DP groups (85 cells/group), respectively. Similar to what we observed in chemically induced diabetes (19), the peak shortening amplitude (PS) normalized to CL was significantly reduced in myocytes isolated from BB/DP diabetic hearts. Myocytes under sustained spontaneous diabetes also exhibited significantly prolonged time-to-peak shortening (TPS) and time-to-90% relengthening (TR₉₀) (Fig. 1). The reduced myocyte shortening and prolonged TPS as well as TR₉₀ are associated with significantly reduced maximal velocities of shortening (+dL/dt) and relengthening (dL/dt) (BB/DP 85 ± 6/77 ± 6 vs. BB/DR 118 ± 5/102 ± 6 µm/ms, P < 0.05).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Contractile properties of cardiac ventricular myocytes isolated from BioBreed diabetic-resistant (BB/DR) and spontaneously diabetic-prone (BB/DP) rat hearts. Graphs are representative cell shortening traces (A), peak shortening amplitude (B), time-to-peak shortening (TPS; C), and time-to-90% relengthening (TR90; D). Myocyte shortening and relengthening were recorded at 37°C. Cells were stimulated to contract at 0.5 Hz. Values are means ± SE; n = 85 cells/group (from 7 BB/DR and 7 BB/DP rat hearts). * P < 0.05 vs. BB/DR group.

Cell shortening and relengthening from the never-diabetic BB/DP rat hearts. The genetically predisposed diabetic BB/DP rats usually turn diabetic at ~60 days after birth. Interestingly, a subset of the BB/DP rats (BB/DP/n) never exhibited hyperglycemia throughout the entire course of the current study. The body and heart weights of the BB/DP/n rats were significantly lower than those of the BB/DR rats, associated with renal hypertrophy. However, the heart and liver size are comparable with those of the BB/DR rats (Table 1). The average resting CL of ventricular myocytes isolated from these rat hearts was 121 ± 4 µm (43 cells). These myocytes displayed similar PS (7.4 ± 0.5% CL), TPS (97 ± 5 ms), and TR₉₀ (150 ± 8 ms) (n = 43 cells/group) compared with the respective parameter obtained from BB/DR rat heart myocytes (P > 0.05). These data suggest that the discrepancy in cardiac mechanical dysfunctions seen in BB/DP rats may not be attributed to the genetic backgrounds of the BB/DR and BB/DP rats.

Intracellular Ca²⁺ transients. We used the membrane-permeant form of fura 2 to evaluate the properties of intracellular Ca²⁺ transients in myocytes from BB/DR and BB/DP rats. The time course of the fluorescence signal decay was well described by a single exponential equation, and the time constant () was used as a measure of the rate of decline of free cytoplasmic Ca²⁺. The fluorescence measurements revealed that the resting Ca²⁺ level as well as the increase of Ca²⁺ (peak  resting) were lower, and intracellular Ca²⁺ transients decayed at a much slower rate in myocytes from the BB/DP group compared with those of the BB/DR group (Fig. 2). These results revealed potential abnormalities in cytoplasmic Ca²⁺ handling and clearing mechanisms in heart from spontaneously diabetic rat. The traces in Fig. 2 were chosen to illustrate that diabetes depressed resting Ca²⁺ ratio and prolonged . Myocyte shortenings were also recorded from fura 2-loaded cells but were used for qualitative comparisons only, to avoid potential effects on contraction from intracellular Ca²⁺ buffering by fura 2. 

View larger version (21K): [in this window] [in a new window]   Fig. 2.   A: typical experiments showing intracellular Ca2+-transient properties in myocytes isolated from BB/DR and BB/DP rat hearts [BB/DR, time constant () = 198 ms; BB/DP,  = 225 ms]. Baseline intracellular Ca2+ levels (B) and Ca2+-transient decay rate (; C) in myocytes from BB/DR and BB/DP rat hearts. Values are means ± SE; n = 16 cells/group (from 3 BB/DR and 3 BB/DP rat hearts). * P < 0.05 vs. BB/DR group.

Effect of extracellular Ca²⁺ on myocyte shortening. The effect of extracellular Ca²⁺ on myocyte shortening was examined and is shown in Fig. 3. Increases in extracellular Ca²⁺ concentration from 0.5 up to 3 mM resulted in a positive staircase in myocyte shortening response in both BB/DR and BB/DP groups. Interestingly, the myocytes from the spontaneously diabetic group were less sensitive to intermediate increase (0.5 mM) in extracellular Ca²⁺ compared with those from the BB/DR group, although this reduced responsiveness was not seen when larger increases of Ca²⁺ were imposed. These data may suggest a possible decrease in the myofilament Ca²⁺ sensitivity in spontaneous diabetes, which has been reported in chemically induced diabetes (9).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Ca2+ dose-response curve in myocytes isolated from BB/DR and BB/DP rat hearts. Values are means ± SE; n = 14 cells/group (from 2 BB/DR and 2 BB/DP rat hearts). # P < 0.05 vs. BB/DR group.

Effect of stimulation frequency on myocyte shortening. Rat hearts normally contract at very high frequencies (300 beat/min at 37°C), whereas our baseline studies were conducted at 0.5 Hz. To look for possible derangement of cardiac excitation-contraction (E-C) coupling at higher frequencies, we increased the stimulating frequency up to 5 Hz (300 beat/min) and recorded the steady-state PS. Cells were initially stimulated to contract at 0.5 Hz for 5 min to ensure steady state before the frequency study was commenced. All the recordings were normalized to PS at 0.1 Hz of the same myocyte. Figure 4 shows a negative staircase in PS with increasing stimulating frequency that is essentially identical in both BB/DR and BB/DP groups. Changes in the stimulating frequency from 0.1 to 5 Hz did not affect the prolongation in TPS and TR₉₀ in spontaneously diabetic rats (data not shown). This data suggested that the intracellular Ca²⁺ storage and release is likely preserved in BB diabetes.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Peak shortening amplitude (PS) of ventricular myocyte isolated from BB/DR and BB/DP rat hearts at different stimulus frequencies. Each point represents PS normalized to that of 0.1 Hz. The baseline PS values at 0.1 Hz are 8.5 ± 0.8 and 8.2 ± 0.7% in BB/DR and BB/DP groups, respectively. Values are means ± SE; n = 15 cells/group (from 3 BB/DR and 3 BB/DP rat hearts).

Effect of norepinephrine and KCl on myocyte shortening. To further explore the alteration in cardiac E-C coupling, myocytes were exposed to norepinephrine (1 µM) and KCl (30 mM), and myocyte shortening was examined. Myocytes from the BB/DP group exhibited significantly reduced myocyte shortening capability compared with those from the BB/DR group. KCl produced similar PS in both groups (Fig. 5). These data indicate a depressed -adrenergic response in heart from spontaneously diabetic rats, which may underscore the differential contractile response in the two groups.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effects of norepinephrine (NE; 1 µM) and KCl (30 mM) on cell shortening in BB/DR or BB/DP myocytes. Data are presented as percent change from the respective control value. Values are means ± SE; n = 20 cells/group (from 3 BB/DR and 3 BB/DP rat hearts). # P < 0.05 vs. BB/DR.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This is the first study describing the mechanical dysfunction in diabetic cardiomyopathy of genetic origin at the myocyte level. Our results indicated that ventricular dysfunctions in spontaneous diabetes stem, at least in part, from distinct myocyte defects. The major mechanical abnormalities observed in spontaneously diabetic cardiac myocytes involve depressed cell shortening and prolonged duration of and reduced velocities of both shortening and relengthening. Furthermore, decreased resting intracellular Ca²⁺ level and Ca²⁺-transient decay may likely contribute to impaired mechanical function in myocytes of sustained genetic diabetes.

Impaired mechanical function is an important component of the adverse effects of diabetes on cardiac performance (19, 25). Overt mechanical abnormalities of both chemical (19, 20) and genetic origin (2, 21, 22) are generally observed in diabetic heart, and the severity of the dysfunctions increases with time. Chemically induced diabetes depresses contractility of isolated ventricular myocytes in a manner similar to that in whole heart preparations (6, 19). Depressed systolic left ventricular pressure and change in left ventricular pressure over time (±dP/dt) have also been reported in severe BB diabetes (2). This is supported by the observation from the current study that myocyte shortening (PS) is significantly reduced in heart from spontaneously diabetic rats. The mechanism of action may be related to decreased cardiac myosin Ca²⁺-ATPase activity, which has been reported in both chemically induced (6, 18) and genetic BB (15) diabetes. Contractility may also be compromised by diabetes because ATP production is depressed, in part, because of decreases in mitochondrial respiration and pyruvate dehydrogenase activity (18).

The most prominent effect of diabetes on cardiac function is abnormal contraction and relaxation. Characteristics of abnormal function include prolonged duration and reduced rate of ventricular contraction and relaxation as measured in intact working hearts (4), isolated papillary muscle (6, 20), and isolated ventricular myocytes (19) in the case of chemically induced diabetes and in working heart of genetic diabetes (2). Similar to the observation in myocytes from chemically induced diabetes, our current study revealed that the duration of shortening (TPS) and relengthening (TR₉₀) was prolonged in sustained spontaneous diabetes, associated with slowed maximal rate of shortening and relengthening (±dL/dt). Several factors have been suggested to contribute to the prolonged duration of contraction and relaxation. Depressed rate of shortening has been associated with diabetes-induced shifts in contractile protein isoforms, such as the redistribution of myosin isozymes from the fast type (V₁) to the slow type (V₃) in both chemically induced (5) and spontaneous diabetes (1). Diabetes also significantly depresses myofilament Ca²⁺ sensitivity, without appreciable changes in maximum-developed tension in chemically skinned papillary muscles (12) and as more recently reported in skinned, isolated ventricular myocytes (9). Interestingly, a switch of myosin heavy-chain isoform from V_I to V₃ (induced by hypothyroidism) has been recently shown to directly lead to reduction in Ca²⁺ sensitivity in cardiac myocytes (16). In this study, the steady-state myocyte shortening in response to increases in extracellular Ca²⁺ concentration in heart from spontaneously diabetic rats is clearly diminished at 1 mM Ca²⁺ compared with that of the nondiabetic group. The decrease of myocyte shortening in diabetes may be attributable to reduced myofilament Ca²⁺ responsiveness/sensitivity, which has been reported in diabetic heart (9). The reduced responsiveness to norepinephrine also suggested depressed -adrenergic receptor number or function in spontaneous diabetes and may contribute to the depressed cardiac contraction in spontaneous diabetes. It is possible that reduced myofilament Ca²⁺ sensitivity may also contribute to the diminished responsiveness to norepinephrine in BB/DP myocytes.

One interesting observation of the current study is that myocytes isolated from the BB/DP/n rats exhibit mechanical function (PS, TPS, and TR₉₀) comparable to those from the BB/DR rats. Therefore, the genetic difference between the BB/DR and BB/DP rats does not appear to be a major factor in the cardiac mechanical dysfunction in diabetes of genetic origin.

Recent evidence has suggested that titin may play a role in the alteration of myocardial function and the development of diabetic cardiomyopathy (11). Titin, an endosarcomeric elastic protein, may function as a bidirectional spring that contributes to both shortening and relengthening of unloaded cardiac myocytes (8). Because titin is likely degraded by trypsin without the protection of protease inhibitor in our cell isolation procedure, the difference in mechanical function between BB/DR and BB/DP myocytes seen in the current study may not be associated with changes of titin. However, it would be intriguing to evaluate the titin abundance in genetically predisposed diabetic heart and determine the role of this giant protein in the altered mechanical function.

Abnormal intracellular Ca²⁺ handling often leads to mechanical dysfunctions in diabetes. We and others have shown that chemically induced diabetes prolongs Ca²⁺ transients associated with E-C coupling on a beat-to-beat basis, which may be responsible for the impaired relaxation (13, 19). The current study revealed that Ca²⁺-transient decay is significantly slower, associated with a low diastolic (resting) Ca²⁺ level, in myocytes from the BB/DP group than those from the BB/DR group, as reported in chemically induced diabetes (13, 19, 20). The slower Ca²⁺-transient decay may be a result of impaired sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) and/or other Ca²⁺-regulating proteins such as Na⁺/Ca²⁺ exchanger (3, 5, 24). Rodrigues et al. (21) demonstrated depressed sarcoplasmic reticulum Ca²⁺ uptake in hearts from BB diabetic rats. Reduced function of SERCA, Na⁺/Ca²⁺ exchanger, or other Ca²⁺-regulating proteins may account for the slow Ca²⁺ clearing that we were able to detect in both the chemically induced and genetic diabetic myocytes. However, the effects of diabetes on the expression and function of these regulating proteins have not been elucidated in intact myocytes.

Although our findings provide the evidence that impaired cardiac E-C coupling in diabetes of genetic origin is likely attributable to changes at the single-cell level, the underlying mechanisms remain to be explored. Given what we know about the role of Ca²⁺-ATPase, SERCA, and Na⁺/Ca²⁺ exchanger in cardiac E-C coupling, the direct impact of spontaneous diabetes on the expression and function of these proteins would be very interesting to investigate.