The role of mitochondria and sarcoplasmic reticulum calcium handling upon reoxygenation of hypoxic myocardium

The mechanism of twitch prolongation of hypoxic myocardium after reoxygenation was studied before and after interventions that affect cellular cyclic nucleotide levels, subcellular calcium handling, or oxygen-derived free radical production/survival. Right ventricular ferret papillary muscles were subjected to two 20-minute periods of hypoxia, each followed by 1 hour of reoxygenation. The first sequence of hypoxia/reoxygenation was done without intervention. Before the second sequence, the pharmacological agent under study was added to the superfusate or the superfusate calcium concentration was increased from 2.5 to 8 mM. Time from peak to 80% decline in twitch tension was measured in the presence and absence of each intervention immediately before each period of hypoxia and after reoxygenation at maximal twitch prolongation. Interventions that affect Ca2+ flux across the sarcolemma (verapamil and 8 mM [Ca2+]o) or agents that affect oxygen free radical production/survival (dimethyl sulfoxide and allopurinol) did not affect twitch prolongation. Pharmacological agents that increase cyclic AMP levels (forskolin and milrinone) or those that inhibit mitochondrial activity (oligomycin B and ruthenium red) attenuated twitch prolongation. Pharmacological agents that decrease cyclic AMP levels (carbachol) or inhibit function of the sarcoplasmic reticulum (ryanodine) augmented twitch prolongation. The effect of mitochondrial inhibitors on intracellular calcium handling during hypoxia and reoxygenation was examined using muscles loaded with the bioluminescent calcium indicator aequorin. Mitochondrial inhibitors abbreviated the calcium transient and maximal twitch prolongation after hypoxia. We conclude that alterations in sarcoplasmic reticulum and mitochondria calcium handling contribute to the prolonged relaxation seen upon reoxygenation of hypoxic myocardium.     

Decrease in calcium-sensing receptor in the progress of diabetic cardiomyopathy

To observe the dynamic expression of calcium-sensing receptor (CaSR) in myocardium of diabetic rats and explore its role in diabetic cardiomyopathy (DCM), 40 male Wistar rats were randomly divided into 4 groups including control, diabetic-4 weeks, diabetic-8 weeks and spermine treatment groups (240 μM of spermine in drinking water). The type 2 Diabetes mellitus (DM) models were established by intraperitoneal injection of streptozotocin (STZ, 30 mg/kg) after high-fat and high-sugar diet for one month. The echocardiographic parameters were measured, cardiac morphology was observed by electron microscope and HE staining. The intracellular calcium concentration ([Ca(2+)](i)) was detected by laser-scanning confocal microscope. Western blot analyzed the expression of CaSR, protein kinase C α(PKC-α) and calcium handling regulators, such as phospholamban (PLN), Ca(2+)-ATPase (SERCA), and ryanodine receptor (RyR). Compared with control group, [Ca(2+)](i) and the expression of CaSR, RyR and SERCA/PLN were decreased, while PKC-α and PLN were significantly increased in a time-dependent manner in diabetic groups. Meanwhile diabetic rats displayed abnormal cardiac structure and systolic and diastolic dysfunction, and spermine (CaSR agonist) could prevent or slow its progression. These results indicate that the CaSR expression of myocardium is reduced in the progress of DCM, and its potential mechanism is related to the impaired intracellular calcium homeostasis.     

Electron probe x-ray microanalysis of sarcolemma and junctional sarcoplasmic reticulum in rabbit papillary muscles: low sodium-induced calcium alterations

This project was undertaken to determine whether electron probe x-ray microanalysis (microprobe analysis) could be utilized to determine the subcellular sites responsible for low sodium-induced calcium accumulation in myocardium. Ultrathin cryosections of rabbit papillary muscles were analyzed using microprobe analysis, and the concentrations (mmol/kg dry wt) of Na, Mg, I, S, Cl, K, and Ca were compared against low sodium (36 mM) and control (139 mM NaCl) muscle groups. Visual resolution of junctional sarcoplasmic reticulum in freeze-dried myocardial sections was achieved, and systematic analysis of junctional sarcoplasmic reticulum and sarcolemma was performed. Myofibrils and mitochondria were also analyzed. Reductions in Na and Cl concentration were measured in virtually all compartments of muscles bathed in low sodium. In addition, low sodium produced a doubling of junctional sarcoplasmic reticulum and sarcolemmal calcium concentrations (p less than 0.01). No significant changes in calcium were observed at other analyzed sites. The increased calcium at the junctional sarcoplasmic reticulum and sarcolemma correlates with (but may not completely account for) the threefold increase in contractility measured after 40 minutes in low sodium concentrations. This work demonstrates that elemental changes associated with the myocardial junctional sarcoplasmic reticulum and sarcolemma are amenable to direct, in situ microprobe analysis and further defines these structures as primary sites of calcium accumulation in low sodium concentrations.     

Hypoxia exacerbates Ca-handling disturbances induced by very low density lipoproteins (VLDL) in neonatal rat cardiomyocytes

It is known that myocardium suffers serious alterations under ischemic conditions such as lipid overloading and electrophysiological alterations. However, it is unknown whether intracellular lipid accumulation and calcium dysfunction share common pathophysiological mechanisms under ischemia. The aims of this study were 1) to analyze the effect of normal and high doses of very low density lipoproteins (VLDL) on lipid content and calcium handling; 2) to investigate whether hypoxia modulates the effect of high VLDL doses; and 3) to identify potentially underlying mechanisms in cardiomyocytes. For this purpose, neonatal rat ventricular myocytes cultures were prepared from hearts of 3-4-day-old rats. High doses of VLDL that induced cholesteryl ester (CE) and triglyceride (TG) accumulation strongly reduced sarco(endo)plasmic reticulum Ca ATPase-2 (SERCA-2) expression, calcium transient amplitude and sarcoplasmic reticulum (SR) calcium loading. Interestingly, hypoxia, by upregulating VLDL-receptor expression (4.5-fold at 16 h) increased CE (1.5-fold) and TG (3-fold) cardiomyocyte content and exacerbated the negative effect of VLDL on SERCA-2 expression. Functionally, the hypoxic exacerbation of VLDL-mediated SERCA-2 downregulation was translated into a stronger decrease in calcium transient amplitude and SR calcium loading in myocytes exposed simultaneously to hypoxia and high VLDL. In conclusion, high VLDL doses alter calcium handling in cardiomyocytes and SERCA-2 play a pivotal role in the hypoxic exacerbation of VLDL-mediated effects on cardiac calcium handling. Potentiation of VLDL's effects under hypoxia is explained, at least in part, by hypoxic upregulation of the expression of VLDL-receptor.     

Hypoxia exacerbates Ca(2+)-handling disturbances induced by very low density lipoproteins (VLDL) in neonatal rat cardiomyocytes

It is known that myocardium suffers serious alterations under ischemic conditions such as lipid overloading and electrophysiological alterations. However, it is unknown whether intracellular lipid accumulation and calcium dysfunction share common pathophysiological mechanisms under ischemia. The aims of this study were 1) to analyze the effect of normal and high doses of very low density lipoproteins (VLDL) on lipid content and calcium handling; 2) to investigate whether hypoxia modulates the effect of high VLDL doses; and 3) to identify potentially underlying mechanisms in cardiomyocytes. For this purpose, neonatal rat ventricular myocytes cultures were prepared from hearts of 3-4-day-old rats. High doses of VLDL that induced cholesteryl ester (CE) and triglyceride (TG) accumulation strongly reduced sarco(endo)plasmic reticulum Ca ATPase-2 (SERCA-2) expression, calcium transient amplitude and sarcoplasmic reticulum (SR) calcium loading. Interestingly, hypoxia, by upregulating VLDL-receptor expression (4.5-fold at 16h) increased CE (1.5-fold) and TG (3-fold) cardiomyocyte content and exacerbated the negative effect of VLDL on SERCA-2 expression. Functionally, the hypoxic exacerbation of VLDL-mediated SERCA-2 downregulation was translated into a stronger decrease in calcium transient amplitude and SR calcium loading in myocytes exposed simultaneously to hypoxia and high VLDL. In conclusion, high VLDL doses alter calcium handling in cardiomyocytes and SERCA-2 play a pivotal role in the hypoxic exacerbation of VLDL-mediated effects on cardiac calcium handling. Potentiation of VLDL's effects under hypoxia is explained, at least in part, by hypoxic upregulation of the expression of VLDL-receptor.     

Transgenic rat hearts overexpressing SERCA2a show improved contractility under baseline conditions and pressure overload

OBJECTIVE: The activity of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) is reduced in the failing myocardium. Therefore, transfer of SERCA2a cDNA is considered as a therapeutical approach. The aim of this study was analysis of the long-term effect of SERCA2a overexpression in normal as well as pressure overload challenged myocardium of transgenic rats. METHODS: Independent transgenic rat lines were established expressing the rat SERCA2a cDNA specifically in the myocardium resulting in increased SERCA2a protein levels by 30-70%. Simultaneous measurements of isometric contraction and calcium transients were carried out in right ventricular papillary muscle preparations. Hemodynamic parameters were measured in hearts of unchallenged rats as well as 10 weeks after pressure overload induced by abdominal aortic banding. RESULTS: Analysis of calcium handling and contractile parameters in isolated right ventricular papillary muscles revealed significant shortening of intracellular calcium transients and half maximal relaxation times (RT(50)). Assessing myocardial contractility in working heart preparations, both transgenic rat lines revealed elevated left ventricular pressure, improved systolic and diastolic parameters, attenuated negative force-frequency relation, and a dose-dependent beta-adrenergic effect. Aortic banding resulted in reduction of left ventricular pressure and worsening of contraction and relaxation parameters with no differences in mortality in both transgenic (+dP/dt 3084+/-96 vs. 3938+/-250 mmHg/s; RT(50) 47.0+/-1.2 vs. 36.7+/-1.4 ms) and wild-type rats (+dP/dt 2695+/-86 vs. 3297+/-122 mmHg/s; RT(50) 53.0+/-1.6 vs. 44.1+/-1.4). SERCA2a overexpressing hearts revealed improved hemodynamic parameters compared to wild-type controls. Acceleration of isovolumetric relaxation characterized by the index Tau was directly correlated to SERCA2a protein concentrations. CONCLUSION: Overexpression of SERCA2a protein results in a positive inotropic effect under baseline conditions remaining preserved under pressure overload without affecting mortality. Therefore therapeutic transfer of SERCA2a may become a potential approach for gene therapy of congestive heart failure. Moreover, transgenic SERCA2a rats will be useful for studies of long-term SERCA2a overexpression in further cardiovascular disease models.     

Changes in connexin 43, metalloproteinase and tissue inhibitor of metalloproteinase during tachycardia-induced cardiomyopathy in dogs

OBJECTIVE: To study changes in connexin, metalloproteinase and tissue inhibitor of metalloproteinase levels during tachycardia-induced cardiomyopathy (TIC). METHODS: Canine models of TIC were established by rapid right atrial pacing at 350-400 beats per min for 8 weeks in 11 dogs, six dogs acted as a sham operation group. Echocardiography, left ventricular pressure and its first derivation with time (positive and negative maximum, dp/dtmax, -dp/dtmax), and intracardiac electrograms were recorded before and after rapid pacing at 1, 4 and 8 weeks. Data were acquired in sinus rhythm. Ultrastructural changes in left ventricular tissue were observed by transmission electron microscope. Connexin 43 (Cx43) levels in the left ventricular myocardium were measured by confocal laser microscopy. The relative abundance of matrix metalloproteinase (MMP-2) and tissue inhibitor of metalloproteinase (TIMP-2) were studied by immunoblotting. RESULT AND CONCLUSIONS: (1) Ventricular dilatation and systolic dysfunction occurred after 1 week of rapid right atrial pacing. (2) There was structural damage to the myofibrils, mitochondria, and the sarcoplasmic reticulum with intercalated disk discontinuity. (3) Levels of Cx43 decreased significantly and gap junction remodelling occurred during TIC. (4) TIC may result from several mechanisms, such as ultrastructural changes or gap junction and matrix remodelling.     

The roles of MMP-2/TIMP-2 in extracellular matrix remodelling in the hearts of STZ-induced diabetic rats

OBJECTIVE: The present study was designed to examine the changes of MMP-2/TIMP-2 in the hearts of streptozotocin-induced diabetic rats and gain insight into their roles in extracellular matrix (ECM) remodelling on an experimental animal model of diabetic cardiomyopathy. METHODS AND RESULTS: Sixteen male Wistar rats were randomly divided into two groups: normal control and diabetic rats. Diabetes mellitus was induced in rats by streptozotocin injection. All rats were fed with standard chow and water ad libitum for 4 weeks. At 4 weeks diabetic rats were associated with a lower body weight (BW) and heart weight (HW) but a higher HW/BW. In the diabetic group, serum MMP-2 level had a tendency to increase but not significantly, while serum TIMP-2 significantly increased. Both the activity and expression of MMP-2 weakened in the hearts of diabetic rats. TIMP-2 gene expression in myocardium enhanced significantly. TIMP-2 protein level in diabetic heart was strengthened slightly but not significantly. VG staining showed a marked deposition of collagen in the diabetic group. Multivariate analysis revealed that total collagen content correlated negatively with the activity and gene expression of MMP-2 in the myocardium, and correlated positively with TIMP-1 mRNA expression. CONCLUSIONS: The decrease in MMP-2 activity and expression and increase in TIMP-2 gene expression in the myocardium of diabetic rats may lead to impairment of collagen degradation and contribute to the matrix deposition in diabetic myocardiopathy. The correlation between the serum level and cardiac expression of TIMP-2 in diabetic rats suggested that serum TIMP-2 level may be a viable marker for early diagnosis of diabetic myocardiopathy.     

rAAV-asPLB transfer attenuates abnormal sarcoplasmic reticulum Ca2+ -ATPase activity and cardiac dysfunction in rats with myocardial infarction

BACKGROUND: Diminished myocardial sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) activity and upregulated phospholamban (PLB) level during cardiac dysfunction, had been reported in many studies. AIMS: The current study was designed to examine the effects of rAAV-antisense phospholamban (asPLB) gene transfer on cardiac function, SERCA expression and activity, as well as PLB expression and phosphorylation (Pser16-PLB), in a rat myocardial infarction (MI) model. METHODS AND RESULTS: Rat MI model was generated by ligating the left anterior descending coronary artery. Four weeks later, left ventricular ejection fraction (LVEF), left ventricular systolic pressure (LVSP), the maximal rates of increase and decrease in intraventricular pressure (+/-dp/dt(max)) were significantly depressed, and left ventricular end diastolic pressure (LVEDP) was increased. Myocardial PLB was markedly increased while both SERCA activity and Pser16-PLB level were decreased. In rAAV-asPLB transfected rats, rAAV-asPLB, which was injected into the myocardium around the infarction area immediately after the coronary artery ligation, effectively attenuated the depression of cardiac function, significantly inhibited the expression of PLB, restored Pser16-PLB level and enhanced myocardium SERCA activity. CONCLUSION: rAAV-asPLB transfer in rats with MI effectively prevented the progression of heart failure.     

Two structurally distinct calcium storage sites in rat cardiac sarcoplasmic reticulum: an electron microprobe analysis study

The elemental composition of subcellular organelles in resting rat papillary muscle was measured by electron probe x-ray microanalysis of cryosections of flash-frozen tissue. Nonmitochondrial electron-dense structures (50-100 nm in diameter) with a phosphorous concentration larger than 375 mmol/kg dry wt were identified in the interfibrillar spaces of the I band region. They were not visible in the proximity of transverse tubules. The sodium, magnesium, phosphorus, sulfur, chlorine, and potassium content of the electron dense structures showed a normal distribution, consistent with a uniform composition of a specific subcellular organelle. However, the distribution of the calcium concentrations in these electron-dense structures was bimodal, suggesting that they are composed of at least two subpopulations. One subpopulation had relatively high calcium (up to 53 mmol/kg dry wt) content with a mean value of 12.5 +/- 1.1 mmol/kg dry wt, while the other one had a relatively low calcium content with a mean value of 2.8 +/- 0.3 mmol/kg dry wt. The mean calcium concentration in the junctional sarcoplasmic reticulum (j-SR) in rat papillary muscle with calcium concentrations larger than 6 mmol/kg dry wt was 14.6 +/- 2.0 mmol/kg dry wt. We propose that the electron-dense structures described above correspond to nonjunctional sarcoplasmic reticulum and that the population containing relatively high calcium concentrations is calsequestrin-containing corbular sarcoplasmic reticulum (c-SR) confined to the I band region, while the population containing relatively low calcium concentrations corresponds to anastomosing regions of the network sarcoplasmic reticulum that lack calsequestrin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutagenesis of Sarcoplasmic Reticulum Ca(2+)-ATPase

Ca(2+)-ATPases of sarco(endo)plasmic reticulum utilize energy derived from hydrolysis of ATP to drive active uptake of calcium ions. Site-directed mutagenesis analysis of Ca(2+)-ATPase structure-function relationships has identified protein domains and single amino acid residues involved in the binding and occlusion of the calcium ions during translocation, as well as amino acid residues crucial to the energy-transducing intramolecular signaling that links events in the catalytic ATP hydrolysis site with rearrangements in the calcium sites. The interaction between the cardiac muscle sarcoplasmic reticulum Ca(2+)-ATPase and the regulatory protein phospholamban, which mediates the activation of Ca(2+) transport by β-agonists, has also been elucidated by mutagenesis.     

辛伐他汀联合卡托普利治疗对糖尿病大鼠心功能和心肌纤维化的影响

目的了解辛伐他汀和卡托普利对糖尿病大鼠心功能和心肌纤维化的影响。方法糖尿病大鼠模型随机分为糖尿病对照（DM）组、辛伐他汀（DM-S）组、卡托普利（DM-C）组、辛伐他汀＋卡托普利（DM-CS）组,分别给药4周后测量左室平均心室峰压（LVSP）、平均心室舒张末压（LVEDP）、平均左室内压最大上升（＋dp/dtmax）和下降速率（-dp/dtmax）等心功能参数。结果 DM-CS组LVSP、＋dp/dtmax、-dp/dtmax及其较正值较DM组明显增加（p〈0.05）,心肌间质纤维化受抑。结论辛伐他汀联合卡托普利可改善糖尿病大鼠心肌间质结构和心脏舒缩功能。     

Effects of phospholamban phosphorylation catalyzed by adenosine 3':5'-monophosphate- and calmodulin-dependent protein kinases on calcium transport ATPase of cardiac sarcoplasmic reticulum

To elucidate the role of 22000-dalton protein phospholamban, a putative regulator of Ca2+-dependent ATPase of cardiac sarcoplasmic reticulum, we examined the relationship between cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban and their effects on ATPase activity and calcium transport of cardiac sarcoplasmic reticulum. Cardiac microsomes were incubated with [gamma-32P]ATP or unlabeled ATP, catalytic subunit of cyclic AMP-dependent protein kinase and/or exogenous calmodulin, and subsequently assayed for ATPase activity and calcium uptake by cardiac sarcoplasmic reticulum. Cyclic AMP-dependent phosphorylation of phospholamban was independent of Ca2+, whereas calmodulin-dependent phosphorylation of phospholamban was dependent on Ca2+ within a range between 0.2 and 50 microM. Cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban occurred independently; when both kinases were operative, the amounts of phosphorylation were additive. Under these conditions, the phosphoproteins formed by cyclic AMP- and calmodulin-dependent protein kinases electrophoretically migrated as 11000-dalton components when sodium dodecyl sulfate-solubilized phosphoproteins were boiled prior to polyacrylamide gel electrophoresis. The ATPase activity was stimulated by either cyclic AMP- or calmodulin-dependent phosphorylation of phospholamban at Ca2+ concentrations up to 2 microM. The extents of stimulation of ATPase activity were additive when both types of phosphorylation were functional. Calcium uptake was similarly augmented by cyclic AMP- and/or calmodulin-dependent phosphorylation of phospholamban. These results indicate that Ca2+-dependent ATPase and calcium transport of cardiac sarcoplasmic reticulum are regulated by phospholamban phosphorylation catalyzed by cyclic AMP- and calmodulin-dependent protein kinases, thus suggesting a dual role of phospholamban in active calcium transport.     

Diminished expression of sarcoplasmic reticulum Ca(2+)-ATPase and ryanodine sensitive Ca(2+)Channel mRNA in streptozotocin-induced diabetic rat heart

The diabetic heart has an abnormal intracellular calcium ([Ca(2+)]i) metabolism. However, the responsible molecular mechanisms are unclear. The present study aimed to investigate mRNAs expressed in the proteins which regulate heart [Ca(2+)]i metabolism in streptozotocin (STZ)-induced diabetic rats. Expression of sarcoplasmic reticulum Ca(2+)-adenosine triphosphatase (SR Ca(2+)-ATPase) mRNA was significantly less in the heart 3 weeks after STZ injection than that in the age-matched controls. Together with the down-regulation of SR Ca(2+)-ATPase, expression of ryanodine sensitive Ca(2+)channel (RYR) mRNA was also decreased 12 weeks after STZ injection. Insulin supplementation fully restored the decreased mRNAs expression of SR Ca(2+)-ATPase and RYR. The diminished expression and restoration with insulin supplementation of SR Ca(2+)-ATPase was further confirmed at the protein level. In contrast, expression of mRNAs coding the L-type Ca(2+)channel, Na(+)-Ca(2+)exchanger, or phospholamban were not affected 3 or 12 weeks after STZ injection. These results can be taken to indicate that the down-regulation of SR Ca(2+)-ATPase and RYR mRNAs is a possible underlying cause of cardiac dysfunction in STZ-induced diabetic rats.     

Inotropic reactions of myocardium of patients with ischemic heart disease and rats during use of amiodarone

AIM: To study effect of amiodarone on inotropic reactions of myocardium of patients with ischemic heart disease and rats. Methods and RESULTS: Excitability of rat myocardium has been found to be higher than that of myocardium of patients. Sarcoplasmic reticulum of myocardium of rats has greater functional reserve, therefore rat muscle preparations respond to extrasystolic stimuli with postextrasystolic potentiation. Perfusion with a solution containing amiodarone caused diminution of amplitude of extrasystolic response of muscular strips both from rats and patients. Amiodarone augmented ability of sarcoplasmic reticulum to accumulate Ca ions as it prevented decrease of postextrasystolic contraction of human myocardium and brought about postextrasystolic potentiation of muscular strips of rat myocardium. CONCLUSION: Amiodarone affected intracellular homeostasis of calcium ions in cardiomyocytes of both rats and patients. Amiodarone lowered myocardial excitability and facilitated ability of cardiomyocyte sarcoplasmic reticulum to accumulate calcium ions.     

Ultrastructure of heart muscle in short-term diabetic rats: influence of insulin treatment

Summary The ultrastructure of myocardium was examined in short-term diabetic rats. Morphometric analysis showed the volume of myocytic mitochondria, sarcoplasmic reticulum and lipid droplets to be significantly increased compared with those of control animals. Further measurements of mitochondria and sarcoplasmic reticulum indicated that the augmentation of these compartments was accountable by the enlargement of pre-existing mitochondria, which were swollen, and of pre-existing tubules of sarcoplasmic reticulum, the lumen of which was dilated. After insulin treatment the morphological changes were returned to normal which indicates that they were not due to the toxic effect of streptozotocin but were caused by the diabetic state per se. This suggestion is further supported by the finding that experimentally induced metabolic acidosis without diabetes did not cause any morphologically detectable changes in the heart muscle. It is concluded that short-term diabetes in the rat causes mitochondrial swelling, dilatation of sarcoplasmic reticulum and accumulation of lipid in cardiac myocytes, and that these changes are preventable with insulin treatment. We suggest that insulin may have an important role in the maintenance of metabolism in heart muscle.     

Aerobic interval training enhances cardiomyocyte contractility and Ca2+ cycling by phosphorylation of CaMKII and Thr-17 of phospholamban

Cardiac adaptation to aerobic exercise training includes improved cardiomyocyte contractility and calcium handling. Our objective was to determine whether cytosolic calcium/calmodulin-dependent kinase II and its downstream targets are modulated by exercise training. A six-week aerobic interval training program by treadmill running increased maximal oxygen uptake by 35% in adult mice, whereupon left ventricular cardiomyocyte function was studied and myocardial tissue samples were used for biochemical analysis. Cardiomyocytes from trained mice had enhanced contractility and faster relaxation rates, which coincided with larger amplitude and faster decay of the calcium transient, but not increased peak systolic calcium levels. These changes were associated with reduced phospholamban expression relative to sarcoplasmic reticulum calcium ATPase and constitutively increased phosphorylation of phospholamban at the threonine 17, but not at the serine 16 site. Calcium/calmodulin-dependent kinase IIdelta phosphorylation was increased at threonine 287, indicating activation. To investigate the physiological role of calcium/calmodulin-dependent kinase IIdelta phosphorylation, this kinase was blocked specifically by autocamtide-2 related inhibitory peptide II. This maneuver completely abolished training-induced improvements of cardiomyocyte contractility and calcium handling and blunted, but did not completely abolish the training-induced increase in Ca(2+) sensitivity. Also, inhibition of calcium/calmodulin-dependent kinase II reduced the greater frequency-dependent acceleration of relaxation that was observed after aerobic interval training. These observations indicate that calcium/calmodulin-dependent kinase IIdelta contributes significantly to the functional adaptation of the cardiomyocyte to regular exercise training.     

Endoplasmic reticulum stress is involved in myocardial apoptosis of streptozocin-induced diabetic rats

Apoptosis plays a critical role in the diabetic cardiomyopathy, and endoplasmic reticulum stress (ERS) is one of the intrinsic apoptosis pathways. Previous studies have shown that the endoplasmic reticulum becomes swollen and dilated in diabetic myocardium, and ERS is involved in heart failure and diabetic kidney. This study is aimed to demonstrate whether ERS is induced in myocardium of streptozotocin (STZ)-induced diabetic rats. We established a type 1 diabetic rat model, used echocardiographic evaluation, hematoxylin-eosin staining, and the terminal deoxynucleotidyl transferase-mediated DNA nick-end labeling staining to identify the existence of diabetic cardiomyopathy and enhanced apoptosis in the diabetic heart. We performed immunohistochemistry, western blot, and real-time PCR to analyze the hallmarks of ERS that include glucose-regulated protein 78, CCAAT/enhancer-binding protein homologous protein (CHOP) and caspase12. We found these hallmarks to have enhanced expression in protein and mRNA levels in diabetic myocardium. Also, another pathway that can lead to cell death of ERS, c-Jun NH(2)-terminal kinase-dependent pathway, was also activated in diabetic heart. Those results suggested that ERS was induced in STZ-induced diabetic rats' myocardium, and ERS-associated apoptosis occurred in the pathophysiology of diabetic cardiomyopathy.     

Impaired SERCA function contributes to cardiomyocyte dysfunction in insulin resistant rats

Ventricular dysfunction in type 2 diabetic patients is becoming apparent early after diagnosis of diabetes, but the cellular mechanisms contributing to this dysfunction are not well established. Our group has recently identified cardiomyocyte dysfunction in diet-induced insulin resistant rats that have not developed type 2 diabetes. The present investigation was designed to determine cellular mechanisms contributing to slowed cardiomyocyte relaxation in sucrose (SU)-fed rats. SU-feeding was used to induce whole-body insulin resistance. After 9-12 weeks on diet, isolated ventricular myocyte shortening/relengthening were slower in SU-fed adult male Wistar rats (42-63%) compared to starch (ST)-fed controls. Cytosolic Ca2+ removal attributable to Na+/Ca2+ exchange (NCX) and to sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) was evaluated with fluo-3/AM. Caffeine-releasable Ca2+ and cytosolic Ca2+ clearing through NCX were normal, whereas Ca2+ uptake by SERCA was significantly slower in SU myocytes (330+/-29 ms) compared to ST cells (253+/-16 ms). Protein levels for SERCA, NCX and phospholamban were not affected by SU-feeding. Manipulating intracellular Ca2+ with various positive inotropic interventions (e.g. post-rest potentiation, isoproterenol) and changes in stimulus frequency demonstrated that mechanical properties can be improved in subsets of myocytes. Thus, we conclude that impaired SERCA activity (with normal protein content) contributes to cardiomyocyte dysfunction in insulin resistant animals, whereas NCX function and expression are normal. These results suggest that subtle changes in Ca2+ regulation which occur prior to overt ventricular dysfunction/failure, may be common to early stages of a number of disorders involving insulin resistance (e.g. diabetes, obesity, syndrome X and hypertension).     

Beneficial effects of angiotensin-converting enzyme inhibition on sarcoplasmic reticulum function in the failing heart of the Dahl rat.

Inhibition of angiotensin-converting enzyme (ACE) retards the process of myocardial remodeling and contractile dysfunction that leads to heart failure. However, the intracellular mechanisms by which ACE inhibition preserves myocardial contractility are largely unclear. Using a model of heart failure induced by hypertension in Dahl salt-sensitive (DS) rats, the mechanisms by which ACE inhibitors (ACEI) exert a beneficial effect on myocardial contractility were studied. Dahl salt-resistant (DR) rats, DS rats not given temocapril (DS/T-), and DS rats treated with temocapril (10 mg/kg per day from 10 to 17 weeks of age, DS/T+) were fed an 8% NaCl diet from 8 to 17 weeks of age (n=8, each group). Echocardiography, hemodynamic measurement, histology, contraction of isolated skinned papillary muscle, and Western blot analysis were carried out. At an elevated final blood pressure similar to that of the DS/T- rats, DS/T+ rats exhibited (1) a decrease in left ventricular (LV) mass associated with decreases in both cardiomyocyte size and interstitial fibrosis; (2) improvement of both systolic and diastolic LV function; and (3) an increase in caffeine contraction after constant Ca(2+)-loading with 8-bromo-cAMP into the sarcoplasmic reticulum (SR) associated with an increase in Ser16-phosphorylated phospholamban, as compared with the DS/T- rats. In addition to inhibition of myocardial remodeling, a restoration of the Ca(2+)-handling ability of the SR by normalized phosphorylated phospholamban may contribute to the improved LV contractile function achieved by chronic treatment with an ACEI.