Preservation of the Positive Lusitropic Effect of β-Adrenoceptors Stimulation in Diabetic Cardiomyopathy

Abstract

BACKGROUND: 

In diabetic cardiomyopathy, diastolic dysfunction results in part from sarcoplasmic reticulum abnormalities affecting both phospholamban and sarcoplasmic reticulum Ca²⁺ uptake (SERCA2a). Consequently, the positive lusitropic effect of β-adrenoceptors stimulation could be altered, and β₃-adrenoceptor over-expression may play a role, as previously demonstrated with an altered positive inotropic effect. In this study, we tested the hypothesis that the β-adrenergic positive lusitropic effect is altered in diabetic cardiomyopathy, and that β₃-adrenoceptor over-expression is involved.

METHODS: 

β-adrenergic responses were investigated in vivo (dobutamine-echocardiography) and in vitro (papillary muscle preparation) in healthy and diabetic rats killed 4 (4W) and 12 (12W) wk after IV streptozotocin injection. The effect of β₃-adrenoceptor pathway inhibition by S-cyanopindolol (selective β₃-adrenoceptor antagonist) or by N^G-nitro-l-arginine-methyl-ester (nonselective nitric oxide synthase inhibitor) on the lusitropic response to isoproterenol (nonselective β-adrenoceptors agonist) was studied in vitro. Western blots were performed to quantify the protein expressions of β₁- and β₃-adrenoceptors, phospholamban, and SERCA2a. Data are presented as mean percentages of baseline ± sd.

RESULTS: 

Despite the increased phospholamban/SERCA2a protein ratio and documented diastolic dysfunction, the positive lusitropic effect of β-adrenoceptors stimulation was preserved in vivo (dobutamine) and in vitro (isoproterenol) in 4W and 12W diabetic, compared with healthy, rats. The β₃-adrenoceptor was up-regulated whereas β₁-adrenoceptor was down-regulated in 4W and 12W diabetic, compared with healthy, rats. Nevertheless, S-cyanopindolol or N^G-nitro-l-arginine-methyl-ester had no lusitropic effect.

CONCLUSIONS: 

The positive lusitropic effect of β-adrenoceptor stimulation was preserved in diabetic cardiomyopathy. β₃-adrenoceptor over-expression does not seem to affect this process.

In Brief

IMPLICATIONS: The positive lusitropic effects of β-adrenoceptor stimulation are preserved in diabetic cardiomyopathy and are not affected by β₃-adrenoceptors over-expression.

Diabetes mellitus affects 170 million individuals worldwide and it is a growing public health problem.¹ Perioperative cardiovascular instability occurs frequently in diabetic patients increasing the perioperative use of vasoactive drugs.² Further, diabetes increases the risk for perioperative morbidity and mortality.¹ It is associated with cardiomyopathy that occurs in 60% of even well-controlled type II diabetic patients, independently of coronary artery disease, cardiac valvular disease, or hypertension.¹ Diabetic cardiomyopathy results from a variety of alterations involving the sarcoplasmic reticulum affecting both phospholamban and sarcoplasmic reticulum Ca²⁺ uptake (SERCA2a), calcium channels, intracellular calcium metabolism, sodium-calcium exchange, mitochondria, and contractile proteins.¹ These abnormalities lead to diastolic dysfunction, which may predispose to increased cardiac filling pressures and perioperative congestive heart failure.¹

An increase in sympathetic nervous system activation is an important mechanism for maintaining cardiac output. Stimulation of β₁- and β₂-adrenoceptors induces a positive inotropic effect resulting from cyclic adenosine monophosphate (cAMP) production and protein kinase A (PKA) activation. The magnitude of this positive inotropic effect can be affected by the negative inotropic effect of β₃-adrenoceptor, whose stimulation is mediated through a nitric oxide pathway,^1,3–5 leading to the double activation of phosphodiesterases and protein kinase G. Once activated, the first enzyme increases the catabolism of the produced cAMP whereas the second decreases PKA activity.⁴ While β₃-adrenoceptor expression is minimal in the healthy heart, the distribution of β-adrenoceptors is markedly modified in diabetic cardiomyopathy: β₁- and β₂-adrenoceptors are down-regulated^3,6,7 whereas the β₃-adrenoceptor is up-regulated.^3,8 This redistribution allows a markedly altered positive inotropic effect of β-adrenoceptors stimulation.^3,9

The positive lusitropic effect of β-adrenoceptors stimulation also contributes to the magnitude of its positive inotropic effect.^9–11 Activated PKA phosphorylates different proteins such as phospholamban, an inhibitor of sarcoplasmic reticulum Ca²⁺ ATPase pump (SERCA2a). Phosphorylated phospholamban increases Ca²⁺ sarcoplasmic reticulum uptake by increasing the affinity for Ca²⁺ of SERCA2a.⁴ The increased Ca²⁺ uptake allows for an increase in the “Ca²⁺ released” from the sarcoplasmic reticulum during stimulation, increasing inotropy. In this study, we tested the hypothesis that the positive lusiotropic effect of β-adrenergic stimulation is altered in diabetic cardiomyopathy and, furthermore, that β₃-adrenoceptor over-expression contributes to this altered response.

METHODS

Animal care was in accordance with the Guiding Principles in the Care and Use of Animals,¹² and the protocol of the study was approved by the Animal Care Committee of our institution under the supervision of authorized researchers (B.R., C.H.) in accordance with the regulations issued by the French Ministry of Agriculture.

Animals

Six-week-old male Wistar rats (Charles River, Saint Germain sur l'Arbresle, France) were tagged with sequential numbers and then randomized into four groups: two age-matched healthy groups and two diabetic groups. Diabetes mellitus was induced with streptozotocin, 65 mg/kg IV (Sigma Chemical, L'Isle d'Abeau Chesnes, France). Diastolic dysfunction has been shown to develop seven days after streptozotocin injection.¹³ Because diabetes induces a progressive alteration of sarcoplasmic reticulum proteins until 5–8 weeks after diabetes onset in this model,^13–16 we investigated two groups of diabetic rats: 1 with early diabetic cardiomyopathy 4 wk after streptozotocin injection (diabetic 4W group) and the other with developed diabetic cardiomyopathy 12 wk after diabetes onset (diabetic 12W group).¹⁶ The age-matched healthy rats were killed at this same time. All animals had continuous access to rat chow and were given water ad libitum. Blood glucose level (Glucotrend, Boehringer, Manheim, Germany) was performed every 48 h the first week after streptozotocin injection and then every week to confirm diabetes (i.e., blood glucose level >35 mM). The animals were selected at random before each experiment. At the time of killing, blood samples were withdrawn from diabetic and control rats and centrifuged at 5000g for 15 min. The plasma fraction was collected and stored at −20°C for further determination of glucose and bicarbonate concentration (Cobas Integra 400, Roche Diagnostic, Manheim, Germany), as reported previously.^3,9,17

Transthoracic Echocardiography

Echocardiography was performed using a General Electric Vivid 7 instrument (Aulnay sous Bois, France) equipped with a 8–14 MHz linear transducer during the infusion of dobutamine 3 days before killing to avoid the confounding influence of the intraperitoneal catecholamine infusion on protein expression. Data were transferred online to a computer for analysis (EchoPAC PC version 2.0.x, General Electric, Velizy, Paris, France). During this procedure, the animals were anesthetized with 1%–2% inhaled isoflurane. Left ventricular diameter was measured in the parasternal long-axis and short-axis views using M-mode ultrasound. Left ventricular ejection fraction, using a modified version of Simpson's monoplane analysis, and left ventricular shortening fraction were measured.¹⁸ Left ventricular diastolic variables were derived using pulsed-wave spectral Doppler mitral flow including: early peak velocity (E wave), late filling due to atrial systole or A wave, deceleration time of E wave, and the isovolumic relaxation time. Further measurements obtained included spectral mitral tissue Doppler imaging from apical view (peak early diastolic velocity, E_a). The E/A ratio was also calculated. The high frequency of E and A wave fusion resulting from the rapid rat heart rate during dobutamine infusion did not allow for conventional Doppler measurements as an estimate of left ventricular diastolic function. However, E_a, which is independent of the cardiac loading conditions, was used during dobutamine stimulation as well as the E/E_a ratio, which was used to estimate left ventricular end-diastolic pressure.^19,20 Dobutamine (4 μg/kg) was administered intraperitoneally and measurements were performed when the heart rate was stabilized.³

Isolated Left Ventricular Papillary Muscle

After anesthesia was induced with intraperitoneal injection of pentobarbital sodium, the heart was quickly removed. The whole heart and the left ventricles were dissected and weighed, and the left ventricular papillary muscles were carefully excised and suspended vertically in a 200-mL jacketed reservoir with Krebs-Henseleit bicarbonate buffer solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 1.1 mM KH₂PO₄, 25 mM NaHCO₃, 2.5 mM CaCl₂, and 4.5 mM glucose) and maintained at 29°C with a thermostatic water circulator. Preparations were field stimulated at 12 pulses/min with 5 ms rectangular wave pulses set just above threshold. The bathing solution was insufflated with 95% oxygen and 5% carbon dioxide, resulting in a pH of 7.40. After a 60-min stabilization period, papillary muscles recovered their optimal mechanical performance. The extracellular concentration of Ca²⁺ was decreased from 2.5 to 0.5 mM, because rat myocardial contractility is nearly maximal at 2.5 mM.^9,17 Conventional mechanical variables at the initial muscle length at the apex of the length-active isometric tension curve were calculated from three twitches. The first twitch was isotonic and was loaded with the preload corresponding to the initial muscle length at the apex of length-active isometric tension curve. The second twitch was abruptly exposed to zero load just after the electrical stimulus with a critical damping. The third twitch was fully isometric at the initial muscle length at the apex of the length-active isometric tension curve. We determined the maximum unloaded shortening velocity using the zero-load technique, and time to peak shortening of the twitch with preload only. In addition, the maximum isometric active force normalized per cross-sectional area and the time to peak force was recorded from the isometric twitch.^3,9,17,21 We determined the maximum unloaded shortening velocity using the zero-load technique and maximum shortening and lengthening velocities and time to peak shortening of the twitch with preload only. In addition, from the isometric twitch, we recorded the maximum isometric active force normalized per cross-sectional area, the peaks of the positive and negative force derivative at the apex of the length-active isometric tension curve normalized per cross-sectional area, the time to peak force; and the time to half relaxation. Because changes in the contraction phase induce coordinated changes in the relaxation phase, indexes of contraction-relaxation coupling have been recommended for the study of lusiotropy.^{3,9,17,21–25} The R1 coefficient (maximum shortening velocity/maximum lengthening velocity) is an indicator of the coupling between contraction and relaxation under low load that is independent of inotropic changes. R1 tests sacroplasmic reticulum function.^{3,9,17,21–26} The R2 coefficient (peak of the positive force derivative/peak of the negative force derivative) measures the coupling between contraction and relaxation under high load in a manner that is less dependent on inotropic changes. R2 indirectly reflects myofilaments calcium sensitivity.^{3,9,17,21–26} At the end of the study, the muscle cross-sectional area was calculated from the length and weight of papillary muscle, assuming a density of 1.

β-adrenoceptor stimulation was induced with incremental concentrations of isoproterenol (10⁻⁸ to 10⁻⁴ M), a nonselective β-adrenoceptor agonist, in the presence of phentolamine (10⁻⁶ M), as previously described.^3,9,24,25 To investigate the β₁-adrenoceptor pathway, we studied additional experiments in which papillary muscles from both 4W diabetic and age-matched healthy rats were exposed to forskolin (5 × 10⁻⁵ M), which directly activates adenylate cyclase, or dibutyryl cAMP (cAMP, 5 × 10⁻⁴ M).^3,27 To assess the role of the β₃-adrenoceptor, we studied additional groups in which papillary muscles from 4W diabetic and age-matched healthy rats were exposed to S-cyanopindolol hemifumarate salt (10⁻¹² M), a β₃-adrenoceptor antagonist,^3,28 or N^G-nitro-l-arginine-methyl-ester (10⁻⁵ M), an unspecific nitric oxide synthase inhibitor.³ The total volume of added drugs did not exceed 2% of the bath volume. All drugs were purchased from Sigma Chemical.

Western Blots

Western blots were performed in seven rats from each group with specific antibodies to measure protein expression of β₁- and β₃-adrenoceptors, SERCA2a, and total phospholamban. Because the β₂-adrenoceptor has an insignificant effect in diabetic cardiomyopathy,^3,8 Western blots were performed to quantify β₁- and β₃-adrenoceptor protein expression. Cardiomyocytes were homogenized in Triton X-100 buffer (1% Triton X-100 with: 50 mmol/L Tris-HCl, pH 7.4, 100 mmol/L NaCl, 50 mmol/L NaF, 5 mmol/L EDTA, 40 mmol/L β-glycerophosphate, 0.2 mmol/L ortho-vanadate, 0.1 mmol/L leupeptine, and 0.001 mmol/L aprotinine) for 1 h at 4°C. After centrifuging at 15,000g for 15 min at 4°C, supernatant protein concentrations were measured using the BCA protein assay kit (Pierce, Perbio Sciences, Brebières, France). Fifty micrograms protein per lane was immunoblotted using rabbit polyclonal anti-SERCA2a (1:500, Affinity Bioreagents, Saint Quentin en Yvelines, France), anti-phospholamban (1:1000, Transduction Laboratories, San Jose, CA, USA), anti-β1-adrenoceptor (1:1000, Affinity Bioreagents), and goat polyclonal anti-β3-adrenoceptor (1:1000, Santa Cruz Biotechnology, Le Perray en Yvelines, France) as previously reported.³ All Western blot experiments were quantified using normalization including a standardization of the different gels. The latter was performed by loading a reference sample on every gel and checking that a similar amount of protein was loaded by measurement of total protein level present on the membrane colored by S-Ponceau. Accordingly, all results were normalized with a link (actin) and the amounts of protein transferred on the membrane were based on these corrected values. Western blot using a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase, was performed and validated to ensure that there was no variation in protein gel loading.

Statistical Analysis

Data are expressed as mean ± sd. Comparison of two means was performed using the Student's t-test and comparison of several means was performed using analysis of variance and Newman-Keuls test. All P values were two-tailed and a P < 0.05 was considered significant. Statistical analysis was performed using NCSS 2001 software (Statistical Solutions Ltd., Cork, Ireland). Concentration-response curves were determined by fitting the data to the Hill sigmoid pharmacological model according to the following equation:

in which Eff_o is the observed effect, Eff_max the maximum effect, C₅₀ the concentration that results in 50% of Eff_max, C the studied concentration, and n the Hill coefficient.⁹ Iterative nonlinear regression curve fitting was used to obtain the best fit (Matlab 1.2c software; The MathWorks, South Natick, MA).

RESULTS

We studied 28 healthy (twenty 4W healthy and eight 12W healthy rats) and 34 diabetic rats (twenty-six 4W diabetic and eight 12W diabetic rats). In diabetic rats, the mortality increased and the difficulty of removing the papillary muscles was enhanced, as previously described.^3,9,17 In this study, 3 diabetic rats died before the expected date (2 in 4W and 1 in 12W groups). In contrast, no healthy rat died during this period. Moreover, 10 papillary muscles were not stable in the diabetic groups (8 in 4W and 2 in 12W groups) compared with only four papillary muscles in healthy groups. Because of the similarity of baseline values in the two control groups (data not shown), all healthy control rats were pooled into one group to compare them with 4W and 12W diabetic rats.

Physical characteristics, blood chemistry results, baseline echocardiography, and left ventricular papillary muscles variables are listed in Table 1. Both 4W and 12W diabetic rats had significantly lower body weight and heart weight than healthy rats, without a significant difference in the heart weight to body weight ratio. Blood glucose levels increased five-fold in diabetic compared with healthy rats. Serum bicarbonate levels were not significantly different between groups, suggesting that ketoacidosis did not occur. Heart rate was slower in the 12W diabetic compared with healthy or 4W diabetic rats (P < 0.05). Systolic function was preserved in 4W and in 12W diabetic rats, as determined with echocardiography in whole animals compared with their control group. Transmitral Doppler findings were consistent with diastolic dysfunction in the two diabetic groups as shown by decreased deceleration time as well as both increased isovolumic relaxation time and E/A ratio. E_a decreased in 12W diabetic compared with healthy rats. Estimates of left ventricular end-diastolic pressure increased in the two diabetic groups compared with healthy group, as shown by the E/E_a ratio. In parallel, maximum unloaded shortening velocity, maximum shortening and lengthening velocities, peaks of the positive and negative force derivative decreased in diabetic rats whereas active force was preserved. Prolongation of both time to peak shortening and time to peak force demonstrated the prolongation of the duration of contraction in diabetic rats. Under isotonic conditions, the relaxation-contraction coupling variable R1 was lower in 4W diabetic rats than in healthy rats and was not further modified in diabetic 12W rats. In isometric conditions, the relaxation-contraction coupling variable R2 was not significantly different between healthy, diabetic 4W, and diabetic 12W rats.

Table 1: Physical Characteristics, Baseline Echocardiography, and Left Ventricular Papillary Muscles Variables in Healthy, 4- and 12-Week-Old Diabetic Rats (4W and 12W)

In Vivo Inotropic and Lusitropic Responses to β-Adrenergic Stimulation

Responses to dobutamine infusion in the whole animal experiments are listed in Table 2. Heart rate increased from baseline in healthy rats, but not in 4W and 12W diabetic groups. Dobutamine increased left ventricular ejection and shortening fractions in each group, but the magnitude of this effect was less in 4W and 12W diabetic groups compared with healthy groups. Dobutamine infusion resulted in an increase in E_a and a decrease in E/E_a ratio in diabetic and control animals indicating enhanced lusitropy and decreased estimated left ventricular end-diastolic pressure, respectively.

Table 2: Inotropic and Lusitropic Response to β-Adrenoceptor Stimulation (Dobutamine 4 μg/kg) in Healthy and Diabetic Rats in Vivo Using Echocardiography

In Vitro Lusitropic Responses to β-Adrenergic Stimulation

Lusitropic responses to varying concentrations of isoproterenol in the papillary muscle preparations including in the presence of β₃-adrenergic receptor antagonist S-cyanopindolol and NOS antagonist L-NAME are listed in Table 3 and Figures 1 and 2. There was no difference in R1 between healthy, 4W, and 12W diabetic rats (61% ± 13%, 64% ± 9%, and 64% ± 10% of baseline value, respectively, P = 0.78) and R2 (85% ± 8%, 80% ± 12%, and 80% ± 8% of baseline value, respectively, P = 0.30) suggesting preserved lusitropic effects of β-adrenoceptors stimulation under isotonic and isometric conditions.

Table 3: Effects of S-Cyanopindolol and N^G-nitro-l-arginine-methyl-ester (L-NAME) on the Lusitropic Responses to β-Adrenoceptor Stimulation (Isoproterenol) in Healthy and Four-Week-Old Diabetic Rats

Figure 1.: Lusitropic response to β-adrenoceptors stimulation under isotonic conditions in healthy (Panel A) and 4 week old diabetic rats (4W Diabetic) (Panel B). Number of papillary muscles was 8 for each group except the healthy control group which involved 16 papillary muscles. R1: ratio of maximum shortening velocity (_maxVc) to maximum lengthening velocity (_maxVr), tested the lusitropic effect under isotonic conditions. Cyanopindolol (β₃-adrenoceptor antagonist) and N^G-nitro-l-arginine-methyl-ester (L-NAME) (nonspecific nitric oxide synthase inhibitor) did not modify the positive lusitropic effect of β-adrenoceptor stimulation in 4W and 12W diabetic rats when compared with healthy rats. Data are mean percentages of baseline values ± sd. NS = no significant difference between groups.

Figure 2.: Lusitropic response to β-adrenoceptors stimulation under isometric conditions in healthy (Panel A) and in 4-wk-old diabetic rats (4W Diabetic) (Panel B). Number of papillary muscles was 8 for each group except the healthy control group which involved 16 papillary muscles. R2 = ratio of the peak of the positive force derivative at L _max normalized per cross-sectional area (+dF/dt) and the peak of the negative force derivative at L _max normalized per cross-sectional area (−dF/dt), tested the lusitropic effect under isometric conditions. Cyanopindolol (β₃-adrenoceptor antagonist) and N^G-nitro-l-arginine-methyl-ester (L-NAME) (nonspecific nitric oxide synthase inhibitor) did not disturb the positive lusitropic effect of β-adrenoceptor stimulation in diabetic rats as compared to healthy rats. Data are mean percentages of baseline values ± sd. NS = no significant difference between groups.

In the presence of the β₃-adrenoceptor antagonist S-cyanopindolol, the positive lusitropic effect of β-adrenoceptors stimulation was modified neither in healthy nor in 4W diabetic rats under isotonic and isometric conditions (Table 3, Figs. 1 and 2). In the presence of the L-NAME, the positive lusitropic effect of β-adrenoceptors stimulation was modified neither in healthy nor in 4W diabetic rats under isotonic and isometric conditions (Table 3, Figs. 1 and 2). S-cyanopindolol or L-NAME alone did not modify R1 or R2 (data not shown).

The comparison of β-adrenoceptors stimulation by isoproterenol, of adenylate cyclase stimulation by forskolin, and the direct effect of cAMP between healthy and diabetic 4W rats is shown Figure 3. Under all these experimental conditions, the positive lusitropic effects under isotonic (Fig. 3A) and isometric conditions (Fig. 3B) were not different between healthy or 4W diabetic groups.

Figure 3.: Comparison of the positive lusitropic effects under isotonic conditions (Panel A) and isometric conditions (Panel B) in the presence of isoproterenol (10⁻⁴ M), dibutyryl cyclic adenosine monophosphate (cAMP) (5 × 10⁻⁴ M), and forskolin (5 × 10⁻⁵ M), in left ventricular papillary muscles from healthy and 4 week old diabetic rats (4W Diabetic). Number of papillary muscles was 8 for each group except the healthy isoproterenol group which involved 16 papillary muscles. R1 = ratio of maximum shortening velocity (_maxVc) to maximum lengthening velocity (_maxVr), tested the lusitropic effect under isotonic conditions; R2 = ratio of the peak of the positive force derivative at L _max normalized per cross-sectional area (+dF/dt) and the peak of the negative force derivative at L _max normalized per cross-sectional area (−dF/dt), tested the lusitropic effect under isometric conditions. Data are mean percentages of baseline values ± sd. NS = no significant difference between healthy and diabetic groups.

Expression of β₁- and β₃-Adrenoceptor Proteins

Western blots and densitometric data reflecting β₁- and β₃-adrenoceptor protein expressions in 4W and 12W diabetic rats compared with age-matched healthy rats were assessed in 7 hearts per groups (Fig. 4). β₁-adrenoceptor was reduced in 4W and 12W diabetic compared with healthy hearts. In contrast, β₃-adrenoceptor increased in 4W and 12W diabetic compared with healthy hearts.

Figure 4.: Representative Western blot and densitometric data reflecting β₁-adrenoceptor proteins expression (Panel A) and β₃-adrenoceptor (Panel B) in 4 week and 12 week old diabetic rats in comparison to healthy rats. Results are expressed as mean ± sd n = 7 in each group. *P < 0.05 vs healthy group.

Expression of SERCA2a and Phospholamban Proteins

Western blots and densitometric data reflecting total phospholamban and SERCA2a proteins expressions (Fig. 5A) and total phospholamban/SERCA2a ratios (Fig. 5B) in seven hearts per groups in diabetic 4W and 12W diabetic rats and compared with age-matched healthy rats were assessed. Compared with control animals, SERCA2a protein expression was decreased in 4W and 12W diabetic rats compared with healthy rats (P < 0.05). In contrast, total phospholamban protein expression was increased in 4W and 12W diabetic rats compared with healthy rats (P < 0.05). The total phospholamban/SERCA2a protein ratio was increased in 4W and 12W diabetic rats compared with controls (P < 0.05). Moreover, the total phospholamban/SERCA2a protein ratio was increased in 12W compared with 4W diabetic rats (P < 0.05).

Figure 5.: Representative Western blot and densitometric data (Panel A) reflecting total phospholamban and SERCA2a proteins expressions and total phospholamban/SERCA2a ratios (Panel B) in 4 week (4W Diabetic) and 12-wk-old diabetic rats (12W Diabetic). Results are expressed as mean ± sd n = 7 in each group. *P < 0.05 vs healthy group; †P < 0.05 vs diabetic 4W group.

DISCUSSION

In the present study, we confirmed, in vivo and in vitro, that myocardial relaxation is impaired in early and in evolved diabetic cardiomyopathy. This diastolic dysfunction appears to result, in part, from the change in phospholamban/SERCA2a distribution.^3,9,14,17 Despite these abnormalities, we observed that the positive lusiotropic effect of β-adrenoceptor stimulation was preserved in early as well as in evolved diabetic cardiomyopathy. β₃-adrenoceptors changes did not interfere with this positive lusiotropic effect.

As previously reported in diabetic cardiomyopathy,^3,15 systolic function was preserved, as shown by unchanged ejection and shortening fractions in vivo, and by preserved active force in vitro. Heart rate is decreased in evolved diabetic cardiomyopathy.¹⁵

Diastolic dysfunction^1,3,29 was confirmed in 4W diabetic rats as shown by an increased E/A ratio as well as decreased deceleration time and E_a wave. These findings are consistent with increased E/E_a ratio demonstrating the enhanced left ventricular filling pressures.¹⁹ The magnitude of these abnormalities was more pronounced in 12W than in 4W diabetic rats, confirming the aggravation of diastolic dysfunction in evolved diabetic cardiomyopathy.¹In vitro, because of the marked slowing of contraction, the contraction-relaxation coupling in isotony, R1, was preserved.^3,9,17 The contraction-relaxation coupling measurement, R2, was not modified in isometry, in agreement with the lack of changes in myofilament Ca²⁺ sensitivity in diabetes.¹⁷ In the diabetic cardiomyocyte, the prolongation of relaxation has been mainly related to an early alteration of the distribution of proteins which regulate cytosolic Ca²⁺ concentration.^1,13,14 Our findings were in agreement because the phospholamban/SERCA2a ratio increased and SERCA2a decreased in the fourth week of diabetes. The fact that SERCA2 decreased earlier than in other studies in which change was observed only in the sixth week of induced diabetes^13,14 may be explained by the two-fold increased blood glucose level in our diabetic animals compared with previous studies.

Lusiotropy plays an important role in the maintenance of cardiac output, yet myocardial relaxation was not evaluated in prior investigations of enhanced inotropy from β-adrenoceptors stimulation.^30–32 Because changes in the contraction phase induce coordinated changes in the relaxation phase, indexes of contraction-relaxation coupling are necessary to appropriately assess lusiotropy.^{3,9,17,21–25} In this study, the enhanced R1 suggested that the positive lusitropic effect of β-adrenoceptors stimulation is preserved. Increased E_a and decreased E/E_a ratio corroborate these findings in vivo.

When a nonspecific β-adrenoceptor agonist is used, β₃-adrenoceptors are also stimulated, which could result in a decrease in β₁-adrenoceptor-mediated cAMP production.⁴ In our study, the lack of a negative lusitropic effect from β₃-adrenoceptor stimulation suggests that the small cAMP production resulting from β₁-adrenoceptor stimulation is sufficient to induce a normal positive lusitropic effect. The concentration of cAMP necessary to induce the maximal positive lusitropic effect is smaller than the concentration necessary to induce the maximal positive inotropic effect.³³ Therefore, whereas the phospholamban/SERCA2a ratio is increased, the phosphorylation of phospholamban by PKA may be sufficient to preserve the lusitropic effect, although the baseline activities of these proteins were decreased. Such important discrepancies between inotropic and lusitropic effects of β₁-adrenoceptor stimulation have been reported in other situations.²⁷ This hypothesis was supported by our findings when cAMP was directly stimulated using dibutyryl cAMP or forskolin. These results demonstrated that the positive lusitropic effects of cAMP were similar between healthy and diabetic hearts, even though the altered positive inotropic effect is partially restored by these drugs.³

The following points should be considered when assessing the clinical relevance of our results. Echocardiography was performed under isoflurane anesthesia, which can interfere with β-adrenergic stimulation in diabetes.⁹ Nevertheless, our results obtained in vivo closely agree with those we obtained in vitro without volatile anesthetics. Further, we did not test the β₃-adrenoceptor influence on the positive lusitropic effect of β-adrenoceptor stimulation in 12W diabetic animals. As the positive lusitropic effect was preserved in the two groups (4W and 12W), and as the β₃-adrenoceptor involvement is early (4 week of diabetes),³ we limited this investigation to the 4W group.

In conclusion, although diastolic dysfunction and an impaired positive inotropic response to β-adrenoceptor stimulation occurs in diabetic cardiomyopathy, we observed, in vivo and in vitro, that the positive lusiotropic effects of β-adrenoceptor stimulation were preserved. Although β₃-adrenoceptors over-expression is responsible for the impaired positive inotropic response to β-adrenoceptor stimulation in diabetes, it does not appear to interfere with the positive lusitropic effect of β-adrenoceptor stimulation.

KACKNOWLEDGMENTS

We thank Dr. David Baker, DM, FRCA (Staff Anesthesiologist, Department of Anesthesiology, CHU Necker-Enfants Malades, Paris), for reviewing the manuscript.