Enhanced phosphoinositide 3-kinase(p110α) activity prevents diabetes-induced cardiomyopathy and superoxide generation in a mouse model of diabetes.

AIMS/HYPOTHESIS: Diabetic cardiomyopathy is characterised by diastolic dysfunction, oxidative stress, fibrosis, apoptosis and pathological cardiomyocyte hypertrophy. Phosphoinositide 3-kinase (PI3K)(p110α) is a cardioprotective kinase, but its role in the diabetic heart is unknown. The aim of this study was to assess whether PI3K(p110α) plays a critical role in the induction of diabetic cardiomyopathy, and whether increasing PI3K(p110α) activity in the heart can prevent the development of cardiac dysfunction in a setting of diabetes. METHODS: Type 1 diabetes was induced with streptozotocin in adult male cardiac-specific transgenic mice with increased PI3K(p110α) activity (constitutively active PI3K [p110α], caPI3K] or decreased PI3K(p110α) activity (dominant-negative PI3K [p110α], dnPI3K) and non-transgenic (Ntg) mice for 12 weeks. Cardiac function, histological and molecular analyses were performed. RESULTS: Diabetic Ntg mice displayed diastolic dysfunction and increased cardiomyocyte size, expression of atrial and B-type natriuretic peptides (Anp, Bnp), fibrosis and apoptosis, as well as increased superoxide generation and increased protein kinase C ß2 (PKCß2), p22 ( phox ) and apoptosis signal-regulating kinase 1 (Ask1) expression. Diabetic dnPI3K mice displayed an exaggerated cardiomyopathy phenotype compared with diabetic Ntg mice. In contrast, diabetic caPI3K mice were protected against diastolic dysfunction, pathological cardiomyocyte hypertrophy, fibrosis and apoptosis. Protection in diabetic caPI3K mice was associated with attenuation of left ventricular superoxide generation, attenuated Anp, Bnp, PKCß2, Ask1 and p22 ( phox ) expression, and elevated AKT. Further, in cardiomyocyte-like cells, increased PI3K(p110α) activity suppressed high glucose-induced superoxide generation and enhanced mitochondrial function. CONCLUSIONS/INTERPRETATION: These results demonstrate that reduced PI3K activity accelerates the development of diabetic cardiomyopathy, and that enhanced PI3K(p110α) activity can prevent adverse cardiac remodelling and dysfunction in a setting of diabetes.

Coenzyme Q10 attenuates diastolic dysfunction, cardiomyocyte hypertrophy and cardiac fibrosis in the db/db mouse model of type 2 diabetes.

AIMS/HYPOTHESIS: An increase in the production of reactive oxygen species is commonly thought to contribute to the development of diabetic cardiomyopathy. This study aimed to assess whether administration of the antioxidant coenzyme Q(10) would protect the diabetic heart against dysfunction and remodelling, using the db/db mouse model of type 2 diabetes. Furthermore, we aimed to compare the efficacy of coenzyme Q(10) to that of the ACE inhibitor ramipril. METHODS: Six-week-old non-diabetic db/+ mice and diabetic db/db mice received either normal drinking water or water supplemented with coenzyme Q(10) for 10 weeks. Endpoint cardiac function was assessed by echocardiography and catheterisation. Ventricular tissue was collected for histology, gene expression and protein analysis. RESULTS: Untreated db/db diabetic mice exhibited hyperglycaemia, accompanied by diastolic dysfunction and adverse structural remodelling, including cardiomyocyte hypertrophy, myocardial fibrosis and increased apoptosis. Systemic lipid peroxidation and myocardial superoxide generation were also elevated in db/db mice. Coenzyme Q(10) and ramipril treatment reduced superoxide generation, ameliorated diastolic dysfunction and reduced cardiomyocyte hypertrophy and fibrosis in db/db mice. Phosphorylation of Akt, although depressed in untreated db/db mice, was restored with coenzyme Q(10) administration. We postulate that preservation of cardioprotective Akt signalling may be a mechanism by which coenzyme Q(10)-treated db/db mice are protected from pathological cardiac hypertrophy. CONCLUSIONS/INTERPRETATION: These data demonstrate that coenzyme Q(10) attenuates oxidative stress and left ventricular diastolic dysfunction and remodelling in the diabetic heart. Addition of coenzyme Q(10) to the current therapy used in diabetic patients with diastolic dysfunction warrants further investigation.

Myocardial oxidative stress contributes to transgenic ß2-adrenoceptor activation-induced cardiomyopathy and heart failure.

BACKGROUND AND PURPOSE: While maintaining cardiac performance, chronic ß-adrenoceptor activation eventually exacerbates the progression of cardiac remodelling and failure. We examined the adverse signalling pathways mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and reactive oxygen species (ROS) after chronic ß2-adrenoceptor activation. EXPERIMENTAL APPROACH: Mice with transgenic ß2-adrenoceptor overexpression (ß2-TG) and non-transgenic littermates were either untreated or treated with an antioxidant (N-acetylcysteine, NAC) or NADPH oxidase inhibitors (apocynin, diphenyliodonium). Levels of ROS, phosphorylated p38 mitogen-activated protein kinase (MAPK), pro-inflammatory cytokines and collagen content in the left ventricle (LV) and LV function were measured and compared. KEY RESULTS: ß2-TG mice showed increased ROS production, phosphorylation of p38 MAPK and heat shock protein 27 (HSP27), expression of pro-inflammatory cytokines and collagen, and progressive ventricular dysfunction. ß2-adrenoceptor stimulation similarly increased ROS production and phosphorylation of p38 MAPK and HSP27 in cultured cardiomyocytes. Treatment with apocynin, diphenyliodonium or NAC reduced phosphorylation of p38 MAPK and HSP27 in both cultured cardiomyocytes and the LV of ß2-TG mice. NAC treatment (500 mg·kg⁻¹ ·day⁻¹) for 2 weeks eliminated the up-regulated expression of pro-inflammatory cytokines and collagen in the LV of ß2-TG mice. Chronic NAC treatment to ß2-TG mice from 7 to 10 months of age largely prevented progression of ventricular dilatation, preserved contractile function (fractional shortening 37 ± 5% vs. 25 ± 3%, ejection fraction 52 ± 5% vs. 32 ± 4%, both P < 0.05), reduced cardiac fibrosis and suppressed matrix metalloproteinase activity. CONCLUSION AND IMPLICATIONS: ß2-adrenoceptor stimulation provoked NADPH oxidase-derived ROS production in the heart. Elevated ROS activated p38 MAPK and contributed significantly to cardiac inflammation, remodelling and failure.

Knockout of beta(1)- and beta(2)-adrenoceptors attenuates pressure overload-induced cardiac hypertrophy and fibrosis.

BACKGROUND AND PURPOSE: The role of beta-adrenoceptors in heart disease remains controversial. Although beta-blockers ameliorate the progression of heart disease, the mechanism remains undefined. We investigated the effect of beta-adrenoceptors on cardiac hypertrophic growth using beta(1)- and beta(2)-adrenoreceptor knockout and wild-type (WT) mice. EXPERIMENTAL APPROACH: Mice were subjected to aortic banding or sham surgery, and their cardiac function was determined by echocardiography and micromanometry. KEY RESULTS: At 4 and 12 weeks after aortic banding, the left ventricle:body mass ratio was increased by 80-87% in wild-type mice, but only by 15% in knockouts, relative to sham-operated groups. Despite the blunted hypertrophic growth, ventricular function in knockouts was maintained. WT mice responded to pressure overload with up-regulation of gene expression of inflammatory cytokines and fibrogenic growth factors, and with severe cardiac fibrosis. All these effects were absent in the knockout animals. CONCLUSION AND IMPLICATIONS: Our findings of a markedly attenuated cardiac hypertrophy and fibrosis following pressure overload in this knockout model emphasize that beta-adrenoceptor signalling plays a central role in cardiac hypertrophy and maladaptation following pressure overload.

Effects of ageing on the activation metabolism of rat papillary muscles.

1. A myothermic technique has been used to investigate the mechanics and energetics of left ventricular papillary muscles from 6-, 15-, 22- and 27-32-month-old Sprague-Dawley rats. 2. There was a significant increase in the left ventricular mass to body mass (LVM:BM) ratio in the senescent. 27-32-month-old group of animals compared with the younger animals (P<0.05). 3. The maximum stress developed in the senescent groups was reduced by almost 40% in comparison with the stress developed by the 6-, 15- and 22-month-old groups (P<0.001). The mean rise time, half relaxation time and half width were increased significantly (P<0.05) in the 22-month-old group but, unexpectedly, this effect was not seen in the senescent group. 4. Heat production per beat versus total stress relationships were obtained in two different ways to determine the magnitude of the activation heat and the isometric economy (given by the slope of the relationship). The activation heat was not significantly different between groups with either method, but there was a significant increase (P<0.001) in the economy with which stress was developed in the senescent group in comparison with the 6- and 15-month-old groups. 5. A combination of forskolin (2.5-6.5 micromol/L) and high Ca2+ (7.5 mmol/L) was used to increase the energy usage per beat. In the 6- and 15-month-old groups, these agents caused a four-fold increase in the activation heat magnitude compared with a less than two-fold increase in the 22-month-old and senescent groups (P<0.001). There was no effect of forskolin/high calcium on the slope of the heat:total stress relationship. 6. The data suggest that, under conditions known to increase cardiac contractility, there is a reduced ability to cycle calcium in the 22-month-old and senescent groups relative to the young adult 6-month-old and adult 15-month-old groups.

Rescue of contractile parameters and myocyte hypertrophy in calsequestrin overexpressing myocardium by phospholamban ablation.

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed cardiac contractile parameters, low Ca(2+)-induced Ca(2+) release from sarcoplasmic reticulum (SR) and cardiac hypertrophy in transgenic mice. To test the hypothesis that inhibition of phospholamban activity may rescue some of these phenotypic alterations, the calsequestrin overexpressing mice were cross-bred with phospholamban-knockout mice. Phospholamban ablation in calsequestrin overexpressing mice led to reversal of the depressed cardiac contractile parameters in Langendorff-perfused hearts or in vivo. This was associated with increases of SR Ca(2+) storage, assessed by caffeine-induced Na(+)-Ca(2+) exchanger currents. The inactivation time of the L-type Ca(2+) current (I(Ca)), which has an inverse correlation with Ca(2+)-induced SR Ca(2+) release, and the relation between the peak current density and half-inactivation time were also normalized, indicating a restoration in the ability of I(Ca) to trigger SR Ca(2+) release. The prolonged action potentials in calsequestrin overexpressing cardiomyocytes also reversed to normal upon phospholamban ablation. Furthermore, ablation of phospholamban restored the expression levels of atrial natriuretic factor and alpha-skeletal actin mRNA as well as ventricular myocyte size. These results indicate that attenuation of phospholamban function may prevent or overcome functional and remodeling defects in hypertrophied hearts.

Effects of aging on the work output and efficiency of rat papillary muscle.

OBJECTIVES: This study aimed to investigate the effect of aging on the work output and efficiency of rat papillary muscle. METHODS: The mechanical and energetic properties of left ventricular papillary muscle preparations isolated from 6-, 15-, and 27- to 32-month-old Sprague-Dawley rats were measured in myothermic experiments at 27 degrees C at a stimulus frequency of 0.167 Hz. RESULTS: We found that the basal metabolism measured in quiescent papillary muscles was significantly reduced in the 27- to 32-month-old group (4.9 mWg(-1) compared to 7.7 and 7.0 mWg(-1) in the 6- and 15-month groups). In isotonic experiments, the work output (at a range of afterloads) was significantly depressed for the 27- to 32-month group being only 52% of the work output of the 6-month group. This outcome was due to a decrease in both the extent of muscle shortening only, 66% of 6- and 15-month data, and in the maximum force developed. The reduced work was accompanied by a parallel decrease in energy consumption (enthalpy) and hence, the net mechanical efficiency (work/active enthalpyx100%) was not altered. A force-length- area (FLA) analysis was applied to the isotonic data and an energy: FLA regression line was obtained for each preparation. We found that there were no significant differences in either the intercept or slope of the energy: FLA relation with age. Contractile efficiency (39+/-3%) in the 27- to 32-month group was not significantly different to that found in the 6-month (43+/-4%) or 15-month (40+/-3% group). CONCLUSION: There are no changes in the mechanical performance or efficiency of cardiac muscle from young (6-month-old) or adult (15-month-old) rats but in the aged and senescent rats (27-32-month-old) there is a pronounced decline in stress development and shortening ability leading to a fall in work output. Mechanical and contractile efficiency however remain unchanged in old age and the data resembles that obtained in pressure overload hypertrophy.

Genetically engineered models with alterations in cardiac membrane calcium-handling proteins.

Regulation of intracellular Ca2+ provides a means by which the strength and duration of cardiac muscle contraction is altered on a beat-to-beat basis. Ca2+ homeostasis is maintained by proteins of the outer cell membrane or sarcolemma and the sarcoplasmic reticulum, which is the major intracellular Ca2+ storage organelle. Recently, genetic engineering techniques designed to induce specific mutations, manipulate expression levels, or change a particular isoform of various membrane Ca(2+)-handling proteins have provided novel approaches in elucidating the physiological role of these gene products in the mammalian heart. This review summarizes findings in murine genetic models with alterations in the expression levels of the sarcolemmal Ca(2+)-ATPase and Na+/Ca2+ exchanger, which move Ca2+ across the cell membrane, and the sarcoplasmic reticulum proteins, which are involved in Ca2+ sequestration (Ca(2+)-ATPase and its regulator, phospholamban), Ca2+ storage (calsequestrin), and Ca2+ release (ryanodine receptor, FK506-binding protein and junctin) during excitation-contraction coupling. Advances in genetic technology, coupled with the development of miniaturized technology to assess cardiac function at multiple levels in the mouse, have added a wealth of new information to our understanding of the functional role of each of these membrane Ca(2+)-handling proteins in cardiac physiology and pathophysiology. Furthermore, these genetic models have provided valuable insights into the compensatory cross-talk mechanisms between the major membrane Ca(2+)-handling proteins in the mammalian heart.

Papillary muscles split in the presence of 2,3-butanedione monoxime have normal energetic and mechanical properties.

A number of studies have used 2,3-butanedione monoxime (BDM) to avoid myocardial damage when small muscle preparations were cut from large hearts. The present study investigates the mechanical and energetic effects of varying muscle cross-sectional area (CSA) by dissection in physiological saline containing BDM. By use of adult rat hearts, three muscle groups were obtained: whole left ventricular papillary muscles (Whole) and left ventricular papillary muscles split longitudinally in the presence of 30 mM BDM, with removal of approximately 10% (BDMSP1) or 40-50% (BDMSP2) of the muscle (5 animals in each group). The isolated muscle preparations were studied at 27 degrees C and stimulated at 0.167 Hz. The Whole and BDMSP1 preparations had comparable CSAs; in isotonically contracting muscles working against a range of afterloads, work, enthalpy (energy use), and mechanical efficiency (work/enthalpy x 100%) were similar for the two groups. In addition, isometric performance [e.g., developed stress (force/CSA), length-tension relationship, and contraction time course] was also similar for the two groups. The thinner BDMSP2 preparations showed an enhanced mechanical performance compared with the Whole and BDMSP1 groups. This outcome was in accordance with data in the literature documenting a negative correlation between stress and CSA. The results suggest that BDM-split and intact papillary muscles of similar CSA have comparable energetic and mechanical properties.

Mechanical and energetic changes in short-term volume and pressure overload of rabbit heart.

The mechanical and energetic consequences of a short-term volume overload (STVOL) hypertrophy and short-term pressure overload (STPOL) hypertrophy have been investigated in rabbits and compared with short-term sham-operated controls (STSOC). Hypertrophy was induced either by creating an aortocaval shunt (volume overload) or by banding the pulmonary artery (pressure overload). Suitable papillary muscles were excised from the hearts 8-10 days after the surgical procedure. At 27 degrees C and a stimulus frequency of 1.0 Hz, peak stress development of the STVOL preparations was significantly reduced from the control group, whereas no significant difference in peak stress development was evident between the STPOL and STSOC groups. Surprisingly, the STPOL preparations displayed pulsus alternans after only 8-10 days of inducing the overload. At steady-state conditions, the isometric 10%-90% rise times, the 90%-10% relaxation times, and the 1/2-widths were not significantly different between the treated and control groups. In isotonically contracting muscles working against a range of afterloads, the enthalpy (energy) and work output of the STVOL and STPOL preparations were depressed compared to the STSOC preparations; the differences were statistically significant for the STVOL group. Due to the parallel change in work and enthalpy, the mechanical efficiency was unaltered. A force-length-area (FLA) analysis, analogue of the pressure-volume-area (PVA) analysis, was applied to the isotonic data of this study. The isotonic enthalpy at the various load levels was plotted against the measured FLA and the data were fitted by linear regression. It was evident that the FLA correlated closely with the energy used. The STVOL and STPOL mean total energy:FLA regression lines lay parallel to but were below the STSOC line, signifying a drop in the activation heat, although this reduction did not achieve statistical significance. It is concluded that significant mechanical and energetic changes are evident after a short-term volume overload although earlier work has shown that these differences are absent at the later, compensated stage of hypertrophy. Changes associated with the pressure overload model suggest a disturbance in calcium regulation: this effect is also seen in long-term pressure overload.