Myocyte Shortening Research Articles

A Gain-Of-Function Mutation in Cardiac Myosin Binding Protein-C Increases Viscoelastic Load and Slows Shortening Velocity in Myocytes from Transgenic Mice

Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein involved in the regulation of cardiac muscle contraction. Effects of cMyBP-C on contraction are thought to be mediated in part by limiting the interactions of actin and myosin to slow myocyte shortening velocity and power output. Although interactions with myosin S2 on the thick filament have been proposed as a way in which cMyBP-C could limit shortening velocity (e.g., by creating a drag force on myosin heads), interactions of cMyBP-C with actin could also account for slowed shortening velocity. For instance, cMyBP-C could create a drag that opposes filament sliding by transiently linking thick and thin filaments together. To explore this possibility we created transgenic mice that express a mutant cMyBP-C with a point mutation, L348P (human L352P), located in a conserved sequence within the regulatory M-domain that increases cMyBP-C binding to actin in vitro. We reasoned that if the mutation also enhanced binding to actin in sarcomeres then shortening velocity would be slowed in myocytes from L348P mice. Results show that transgenic mice expressing the L348P mutation are viable and that L348P cMyBP-C is expressed in sarcomeres. Permeabilized myocytes from transgenic mice showed altered force production including reduced maximal force and enhanced calcium sensitivity of tension. Shortening velocity and power output were significantly reduced whereas passive stiffness and myocyte visco-elasticity were significantly increased. Together these data are consistent with the idea that cMyBP-C creates an internal load in the sarcomere by binding to actin.

Effects of stretch and shortening on gene expression in intact myocardium

Multiple cues have been suggested as the mechanical stimulus for the heart's hypertrophic response. Our work has previously suggested that the amount of cyclic shortening in cardiomyocytes controls myocyte shape and the amount of stretch controls myocyte size. To identify gene expression changes that occur in response to these mechanical perturbations, we used microarray analysis of papillary muscles cultured for 12 h at physiological or reduced levels of cyclic shortening and physiological or reduced mean stretch. Overall, genes related to extracellular matrix (ECM) were surprisingly prominent in our analysis. Connective tissue growth factor was among a small group of genes regulated by the amount of cyclic shortening regardless of the level of mean stretch, and many more ECM genes were regulated by shortening with reduced amounts of stretch. When we compared our results to gene expression data from an in vivo model of pressure overload (PO), which also decreases myocyte shortening, we found the genes that were commonly regulated in PO and our decreased shortening groups were most significantly enriched for ontology terms related to the ECM, followed by genes associated with mechanosensing and the cytoskeleton. The list of genes regulated in PO and our decreased shortening groups also includes genes known to change early in hypertrophy, such as myosin heavy chain 7, brain natriuretic peptide, and myosin binding protein C. We conclude that in intact myocardium, the amount of cyclic shortening may be an important regulator not only of myocyte genes classically associated with hypertrophy but also of ECM genes.

Morphology and Contractility of Cardiac Myocytes in Early Stages of Streptozotocin-Induced Diabetes Mellitus in Rats

Diabetic cardiomyopathy is the leading cause of mortality in type 1 diabetes. Thus study of cardiomyocyte morphology and function during early stages of diabetes using modern analytical methods is of critical importance. Therefore, using confocal microscopy, we determined metric parameters, volumes and contractility, with calcium transients in isolated left-ventricular myocytes at one week after induction of diabetes in rats. Myocyte volume analysis from 3D confocal scans was performed using an automated contour detection algorithm that took the actual shape of the myocytes into account. We showed a significant reduction in myocyte volume in diabetic animals. We also showed a significant reduction in length and width but not in thickness of the myocytes, which suggests disproportional reorganization of the structure of the heart tissue during short-term diabetes. From a functional point of view, we observed a significant decrease in cell shortening at a stimulation frequency of 0.5 Hz. This was accompanied by a decrease in calcium transient amplitude. Together, these data suggest that impaired calcium handling is one of the factors that contributes to the observed decrease in myocyte shortening during early stages of streptozotocin-induced diabetes in rats.

Deletion of the β2-adrenergic receptor prevents the development of cardiomyopathy in mice

Beta adrenergic receptor (β-AR) subtypes act through diverse signaling cascades to modulate cardiac function and remodeling. Previous in vitro studies suggest that β1-AR signaling is cardiotoxic whereas β2-AR signaling is cardioprotective, and may be the case during ischemia/reperfusion in vivo. The objective of this study was to assess whether β2-ARs also play a cardioprotective role in the pathogenesis of non-ischemic forms of cardiomyopathy. To dissect the role of β1 vs β2-ARs in modulating MLP (Muscle LIM Protein) cardiomyopathy, we crossbred MLP−/− with β1−/− or β2−/− mice. Deletion of the β2-AR improved survival, cardiac function, exercise capacity and myocyte shortening; by contrast haploinsufficency of the β1-AR reduced survival. Pathologic changes in Ca2+ handling were reversed in the absence of β2-ARs: peak Ca2+ and SR Ca2+ were decreased in MLP−/− and β1+/−/MLP−/− but restored in β2−/−MLP−/−. These changes were associated with reversal of alterations in troponin I and phospholamban phosphorylation. Gi inhibition increased peak and baseline Ca2+, recapitulating changes observed in the β2−/−/MLP−/−. The L-type Ca2+ blocker verapamil significantly decreased cardiac function in β2−/−MLP−/− vs WT. We next tested if the protective effects of β2-AR ablation were unique to the MLP model using TAC-induced heart failure. Similar to MLP, β2−/− mice demonstrated delayed progression of heart failure with restoration of myocyte shortening and peak Ca2+ and Ca2+ release. Deletion of β2-ARs prevents the development of MLP−/− cardiomyopathy via positive modulation of Ca2+ due to removal of inhibitory Gi signaling and increased phosphorylation of troponin I and phospholamban. Similar effects were seen after TAC. Unlike previous models where β2-ARs were found to be cardioprotective, in these two models, β2-AR signaling appears to be deleterious, potentially through negative regulation of Ca2+ dynamics.

Functional subcellular distribution of β1 - and β2 -adrenergic receptors in rat ventricular cardiac myocytes

β-adrenergic stimulation is a key regulator of cardiac function. The localization of major cardiac adrenergic receptors (β1 and β2) has been investigated using biochemical and biophysical approaches and has led to contradictory results. This study investigates the functional subcellular localization of β1- and β2-adrenergic receptors in rat ventricular myocytes using a physiological approach. Ventricular myocytes were isolated from the hearts of rat and detubulated using formamide. Physiological cardiac function was measured as Ca2+ transient using Fura-2-AM and cell shortening. Selective activation of β1- and β2-adrenergic receptors was induced with isoproterenol (0.1 μmol/L) and ICI-118,551 (0.1 μmol/L); and with salbutamol (10 μmol/L) and atenolol (1 μmol/L), respectively. β1- and β2-adrenergic stimulations induced a significant increase in Ca2+ transient amplitude and cell shortening in intact rat ventricular myocytes (i.e., surface sarcolemma and t-tubules) and in detubulated cells (depleted from t-tubules, surface sarcolemma only). Both β1- and β2-adrenergic receptors stimulation caused a greater effect on Ca2+ transient and cell shortening in detubulated myocytes than in control myocytes. Quantitative analysis indicates that β1-adrenergic stimulation is ∼3 times more effective at surface sarcolemma compared to t-tubules, whereas β2- adrenergic stimulation occurs almost exclusively at surface sarcolemma (∼100 times more effective). These physiological data demonstrate that in rat ventricular myocytes, β1-adrenergic receptors are functionally present at surface sarcolemma and t-tubules, while β2-adrenergic receptors stimulation occurs only at surface sarcolemma of cardiac cells.

Aerobic Interval Training Partly Reverse Contractile Dysfunction and Impaired Ca2+ Handling in Atrial Myocytes from Rats with Post Infarction Heart Failure

BackgroundThere is limited knowledge about atrial myocyte Ca2+ handling in the failing hearts. The aim of this study was to examine atrial myocyte contractile function and Ca2+ handling in rats with post-infarction heart failure (HF) and to examine whether aerobic interval training could reverse a potential dysfunction.Methods and resultsPost-infarction HF was induced in Sprague Dawley rats by ligation of the left descending coronary artery. Atrial myocyte shortening was depressed (p<0.01) and time to relaxation was prolonged (p<0.01) in sedentary HF-rats compared to healthy controls. This was associated with decreased Ca2+ amplitude, decreased SR Ca2+ content, and slower Ca2+ transient decay. Atrial myocytes from HF-rats had reduced sarcoplasmic reticulum Ca2+ ATPase activity, increased Na+/Ca2+-exchanger activity and increased diastolic Ca2+ leak through ryanodine receptors. High intensity aerobic interval training in HF-rats restored atrial myocyte contractile function and reversed changes in atrial Ca2+ handling in HF.ConclusionPost infarction HF in rats causes profound impairment in atrial myocyte contractile function and Ca2+ handling. The observed dysfunction in atrial myocytes was partly reversed after aerobic interval training.

Mathematical modelling of active contraction in isolated cardiomyocytes

We investigate the interaction of intracellular calcium spatio-temporal variations with the self-sustained contractions in cardiac myocytes. A consistent mathematical model is presented considering a hyperelastic description of the passive mechanical properties of the cell, combined with an active-strain framework to explain the active shortening of myocytes and its coupling with cytosolic and sarcoplasmic calcium dynamics. A finite element method based on a Taylor-Hood discretization is employed to approximate the nonlinear elasticity equations, whereas the calcium concentration and mechanical activation variables are discretized by piecewise linear finite elements. Several numerical tests illustrate the ability of the model in predicting key experimentally established characteristics including: (i) calcium propagation patterns and contractility, (ii) the influence of boundary conditions and cell shape on the onset of structural and active anisotropy and (iii) the high localized stress distributions at the focal adhesions. Besides, they also highlight the potential of the method in elucidating some important subcellular mechanisms affecting, e.g. cardiac repolarization.

Theoretical analysis on the relationship between left ventricular energetic efficiency and acute infarct size

Energetic efficiency is an important indicator of cardiac function in acute myocardial infarction. However, the relationship between cardiac energetic efficiency and infarct size is not perfectly elucidated. In this study, the relationship is analysed by means of simulation using a theoretical model of the guinea pig left ventricle. In simulation with varied ratios of infarct area, pressure-volume area (PVA), which is an index of total mechanical energy by ventricular contraction, and myocardial oxygen consumption (MVO2) are calculated for each infarct ratio. Then, change of PVA when MVO2 alters (PVA/MVO2) as a well-known index of energy conversion efficiency is evaluated. In addition, PVA/VO2, which represents a ratio of PVA change to alteration of mean oxygen consumption of myocytes except for infarct myocytes, is introduced as an index for real energetic efficiency. In simulation results, PVA/MVO2 increases but PVA/VO2 decreases as infarct area expands, because with expansion of infarct area PVA decreases but VO2 remains almost unchanged because of larger shortening of myocytes. This implies that the enlargement of shortening of noninfarcted myocyte to compensate for depression of cardiac output is a potential cause of myocardial remodelling.

A Mathematical Model of the Mouse Ventricular Myocyte Contraction

Mathematical models of cardiac function at the cellular level include three major components, such as electrical activity, Ca2+ dynamics, and cellular shortening. We developed a model for mouse ventricular myocyte contraction which is based on our previously published comprehensive models of action potential and Ca2+ handling mechanisms. The model was verified with extensive experimental data on mouse myocyte contraction at room temperature. In the model, we implemented variable sarcomere length and indirect modulation of the tropomyosin transition rates by Ca2+ and troponin. The resulting model described well steady-state force-calcium relationships, dependence of the contraction force on the sarcomere length, time course of the contraction force and myocyte shortening, frequency dependence of the contraction force and cellular contraction, and experimentally measured derivatives of the myocyte length variation. We emphasized the importance of the inclusion of variable sarcomere length into a model for ventricular myocyte contraction. Differences in contraction force and cell shortening for epicardial and endocardial ventricular myocytes were investigated. Model applicability for the experimental studies and model limitations were discussed.

Effects of exercise training on excitation–contraction coupling and related mRNA expression in hearts of Goto-Kakizaki type 2 diabetic rats

Although, several novel forms of intervention aiming at newly identified therapeutic targets are currently being developed for diabetes mellitus (DM), it is well established that physical exercise continues to be one of the most valuable forms of non-pharmacological therapy. The aim of the study was to investigate the effects of exercise training on excitation-contraction coupling and related gene expression in the Goto-Kakizaki (GK) type 2 diabetic rat heart and whether exercise is able to reverse diabetes-induced changes in excitation-contraction coupling and gene expression. Experiments were performed in GK and control rats aged 10-11 months following 2-3 months of treadmill exercise training. Shortening, [Ca(2+)]i and L-type Ca(2+) current were measured in ventricular myocytes with video edge detection, fluorescence photometry and whole cell patch clamp techniques, respectively. Expression of mRNA was assessed in ventricular muscle with real-time RT-PCR. Amplitude of shortening, Ca(2+) transients and L-type Ca(2+) current were not significantly altered in ventricular myocytes from GK sedentary compared to control sedentary rats or by exercise training. Expression of mRNA encoding Tpm2, Gja4, Atp1b1, Cacna1g, Cacnb2, Hcn2, Kcna3 and Kcne1 were up-regulated and Gja1, Kcnj2 and Kcnk3 were down-regulated in hearts of sedentary GK rats compared to sedentary controls. Gja1, Cav3 and Kcnk3 were up-regulated and Hcn2 was down-regulated in hearts of exercise trained GK compared to sedentary GK controls. Ventricular myocyte shortening and Ca(2+) transport were generally well preserved despite alterations in the profile of expression of mRNA encoding a variety of cardiac muscle proteins in the adult exercise trained GK diabetic rat heart.

BKCa++ channel inhibition improves myocardial stunning following hyperkalemic cardioplegic arrest (CP) but not reversible ischemia. Potential role of ROS and GSK3B.

We previously demonstrated, in contrast to pure ischemic injury, that inhibition of BKCa channels improves cardiac contractile recovery following CP. We now investigate the mechanism via which BKCa blockade may be protective during CP. Isolated rat hearts and myocytes were subjected to either in vitro ischemia (30 mins) or hyperkalemic CP (2hrs) followed by reperfusion (30 min). Mitochondrial membrane potential (mMP), ROS generation (n=12), and BKCa‐mediated signaling (n=3) were measured in H9c2 cells with or without CP and the BKCa blocker paxilline (pax). CP and ischemia reduced cardiac contractile function in hearts (n&gt;;6) and myocytes (n&gt;;=15). BKCa blockade increased whole heart recovery and myocyte shortening in CP, but not in ischemic groups. Pax did not affect Ca++ transients in myocytes, or indicators of Ca++ signaling and survival in hearts and H9c2 cells (phospho‐PKA, p‐cTnI, PKA, pAkt, pPLB, p‐sHSP). In H9c2 cells, CP reduced mMP and increased ROS, which was partially blocked by pax. Pax increased phosphorylation of GSK3B in both hearts and cells, independent of changes in Akt phosphorylation. In contrast to protective effects of BKCa openers in the setting of ischemic injury, BKCa channel blockers may improve cardiac recovery in the context of hyperkalemic CP. Cardioprotective mechanisms associated with BKCa blockade include normalized ROS and mMP, and increased GSK3B phosphorylation.

Exercise training prior to myocardial infarction attenuates cardiac deterioration and cardiomyocyte dysfunction in rats

OBJECTIVES:The present study was performed to investigate 1) whether aerobic exercise training prior to myocardial infarction would prevent cardiac dysfunction and structural deterioration and 2) whether the potential cardiac benefits of aerobic exercise training would be associated with preserved morphological and contractile properties of cardiomyocytes in post-infarct remodeled myocardium.METHODS:Male Wistar rats underwent an aerobic exercise training protocol for eight weeks. The rats were then assigned to sham surgery (SHAM), sedentary lifestyle and myocardial infarction or exercise training and myocardial infarction groups and were evaluated 15 days after the surgery. Left ventricular tissue was analyzed histologically, and the contractile function of isolated myocytes was measured. Student's t-test was used to analyze infarct size and ventricular wall thickness, and the other parameters were analyzed by the Kruskal-Wallis test followed by Dunn's test or a one-way analysis of variance followed by Tukey's test (p<0.05).RESULTS:Myocardial infarctions in exercise-trained animals resulted in a smaller myocardial infarction extension, a thicker infarcted wall and less collagen accumulation as compared to myocardial infarctions in sedentary animals. Myocardial infarction-induced left ventricular dilation and cardiac dysfunction, as evaluated by +dP/dt and -dP/dt, were both prevented by previous aerobic exercise training. Moreover, aerobic exercise training preserved cardiac myocyte shortening, improved the maximum shortening and relengthening velocities in infarcted hearts and enhanced responsiveness to calcium.CONCLUSION:Previous aerobic exercise training attenuated the cardiac dysfunction and structural deterioration promoted by myocardial infarction, and such benefits were associated with preserved cardiomyocyte morphological and contractile properties.

Vitamin E Ameliorates the Decremental Effect of Paraquat on Cardiomyocyte Contractility in Rats

BackgroundExposure to pesticides and industrial toxins are implicated in cardiovascular disease. Paraquat (PAR) is a toxic chemical widely used as an herbicide in developing countries and described as a major suicide agent. The hypothesis tested here is that PAR induced myocardial dysfunction may be attributed to altered mechanisms of Ca2+ transport which are in turn possibly linked to oxidative stress. The mechanisms of PAR induced myocardial dysfunction and the impact of antioxidant protection was investigated in rat ventricular myocytes.MethodologyForty adult male Wistar rats were divided into 4 groups receiving the following daily intraperitoneal injections for 3 weeks: Group 1 PAR (10 mg/kg), Control Group 2 saline, Group 3 vitamin E (100 mg/kg) and Group 4 PAR (10 mg/kg) and vitamin E (100 mg/kg). Ventricular action potentials were measured in isolated perfused heart, shortening and intracellular Ca2+ in electrically stimulated ventricular myocytes by video edge detection and fluorescence photometry techniques, and superoxide dismutase (SOD) and catalase (CAT) levels in heart tissue.Principal FindingsSpontaneous heart rate, resting cell length, time to peak (TPK) and time to half (THALF) relaxation of myocyte shortening were unaltered. Amplitude of shortening was significantly reduced in PAR treated rats (4.99±0.26%) and was normalized by vitamin E (7.46±0.44%) compared to controls (7.87±0.52%). PAR significantly increased myocytes resting intracellular Ca2+ whilst TPK and THALF decay and amplitude of the Ca2+ transient were unaltered. The fura-2–cell length trajectory during the relaxation of the twitch contraction was significantly altered in myocytes from PAR treated rats compared to controls suggesting altered myofilament sensitivity to Ca2+ as it was normalized by vitamin E treatment. A significant increase in SOD and CAT activities was observed in both PAR and vitamin E plus PAR groups.ConclusionsPAR exposure compromised rats heart function and ameliorated by vitamin E treatment.

Hydralazine and Organic Nitrates Restore Impaired Excitation-Contraction Coupling by Reducing Calcium Leak Associated with Nitroso-Redox Imbalance*

Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1(-/-)) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca(2+) cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1(-/-) compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca(2+) transient (Δ[Ca(2+)]i) responses in NOS1(-/-) cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca(2+)]i across the range of pacing frequencies and increased myofilament Ca(2+) sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca(2+) reuptake, and restored SR Ca(2+) content. HYD and NTG at low concentrations (1 μm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca(2+) leak, HYD improves Ca(2+) cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.

Effects of Nitrosation on Cardiac Myocyte Shortening

In the heart, there are two pathways for nitric oxide (NO) signaling, the classic cGMP-PKG and S-nitrosation of proteins. The nitrosation pathway involves an oxidation of cysteine thiols by nitrosonium NO+, and can therefore be a significant, reversible post-translational modification of proteins. Previous studies using NO donors such as S-nitroso-N-acetylpenicillamine (SNAP) were unable to resolve between to two pathways. We hypothesized that nitrosation is an important post-translational modifier of myocardial contractility. To test this hypothesis, we examined the effects of S-nitroso-L-cysteine (CysNO), a selective nitrosonium donor that is actively transported into the cell, on sarcomere shortening in unloaded mouse cardiac myocytes. Perfusion of cardiac myocytes with CysNO (30 μM) resulted in the reduction of sarcomere length changes by ∼80%. This period of blunted sarcomere length changes was followed by a ∼15 second exponential recovery in sarcomere length. During the recovery phase, the maximal velocities of shortening and re-lengthening were diminished. Our data suggest a possible in vivo role of NO+ in regulating myocardial contractility.

Biocompatibility of Calcined Mesoporous Silica Particles with Ventricular Myocyte Structure and Function

In vivo and in vitro systems were employed to investigate the biocompatibility of two forms of calcined mesoporous silica microparticles, MCM41-cal and SBA15-cal, with ventricular myocytes. These particles have potential clinical use in delivering bioactive compounds to the heart. Ventricular myocytes were isolated from 6 to 8 week male Wistar rats. The distribution of the particles in ventricular myocytes was investigated by transmission electron microscopy and scanning electron microscopy. The distribution of particles was also examined in cardiac muscle 10 min after intravenous injection of 2.0 mg/mL MCM41-cal. Myocyte shortening and the Ca(2+) transient were determined following exposure to 200 μg/mL MCM41-cal or SBA15-cal for 10 min. Within 10 min of incubation at 25 °C, both MCM41-cal and SBA15-cal were found attached to the plasma membrane, and some particles were observed inside ventricular myocytes. MCM41-cal was more abundant inside the myocytes than SBA15-cal. The particles had a notable affinity to mitochondrial membranes, where they eventually settled. Within 10 min of intravenous injection (2.0 mg/mL), MCM41-cal traversed the perivascular space, and some particles entered ventricular myocytes and localized around the mitochondrial membranes. The amplitude of shortening was slightly reduced in myocytes superperfused with MCM41-cal or SBA15-cal. The amplitude of the Ca(2+) transient was significantly reduced in myocytes superperfused with MCM41-cal but was only slightly reduced with SBA15-cal. Overall, the results show reasonable bioavailability and biocompatibility of MCM41-cal and SBA15-cal with ventricular myocytes.

Structure–Activity Studies of RFamide-Related Peptide-1 Identify a Functional Receptor Antagonist and Novel Cardiac Myocyte Signaling Pathway Involved in Contractile Performance

Human RFamide-related peptide-1 (hRFRP-1, MPHSFANLPLRF-NH(2)) binds to neuropeptide FF receptor 2 (NPFF(2)R) to dramatically diminish cardiovascular performance. hRFRP-1 and its signaling pathway may provide targets to address cardiac dysfunction. Here, structure-activity relationship, transcript, Ca(2+) transient, and phospholabeling data indicate the presence of a hRFRP-1 pathway in cardiomyocytes. Alanyl-substituted and N-terminal truncated analogues identified that R(11) was essential for activity, hRFRP-1((8-12)) mimicked hRFRP-1, and [A(11)]hRFRP-1((8-12)) antagonized the effect of hRFRP-1 in cellular and integrated cardiac performance. RFRP and NPFF(2)R transcripts were amplified from cardiomyocytes and heart. Maintenance of the Ca(2+) transient when hRFRP-1 impaired myocyte shortening indicated the myofilament was its primary downstream target. Enhanced myofilament protein phosphorylation detected after hRFRP-1 treatment but absent in [A(11)]hRFRP-1((8-12))-treated cells was consistent with this result. Protein kinase C (PKC) but not PKA inhibitor diminished the influence of hRFRP-1 on the Ca(2+) transient. Molecules targeting this pathway may help address cardiovascular disease.

Shortening and intracellular Ca2+ in ventricular myocytes and expression of genes encoding cardiac muscle proteins in early onset type 2 diabetic Goto–Kakizaki rats

There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus. Cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. Contractile dysfunction, associated with disturbances in excitation-contraction coupling, has been widely demonstrated in the diabetic heart. The aim of this study was to investigate the pattern of cardiac muscle genes that are involved in the process of excitation-contraction coupling in the hearts of early onset (8-10 weeks of age) type 2 diabetic Goto-Kakizaki (GK) rats. Gene expression was assessed in ventricular muscle with real-time RT-PCR; shortening and intracellular Ca(2+) were measured in ventricular myocytes with video edge detection and fluorescence photometry, respectively. The general characteristics of the GK rats included elevated fasting and non-fasting blood glucose and blood glucose at 120 min following a glucose challenge. Expression of genes encoding cardiac muscle proteins (Myh6/7, Mybpc3, Myl1/3, Actc1, Tnni3, Tnn2, Tpm1/2/4 and Dbi) and intercellular proteins (Gja1/4/5/7, Dsp and Cav1/3) were unaltered in GK ventricle compared with control ventricle. The expression of genes encoding some membrane pumps and exchange proteins was unaltered (Atp1a1/2, Atp1b1 and Slc8a1), whilst others were either upregulated (Atp1a3, relative expression 2.61 ± 0.69 versus 0.84 ± 0.23) or downregulated (Slc9a1, 0.62 ± 0.07 versus 1.08 ± 0.08) in GK ventricle compared with control ventricle. The expression of genes encoding some calcium (Cacna1c/1g, Cacna2d1/2d2 and Cacnb1/b2), sodium (Scn5a) and potassium channels (Kcna3/5, Kcnj3/5/8/11/12, Kchip2, Kcnab1, Kcnb1, Kcnd1/2/3, Kcne1/4, Kcnq1, Kcng2, Kcnh2, Kcnk3 and Kcnn2) were unaltered, whilst others were either upregulated (Cacna1h, 0.95 ± 0.16 versus 0.47 ± 0.09; Scn1b, 1.84 ± 0.16 versus 1.11 ± 0.11; and Hcn2, 1.55 ± 0.15 versus 1.03 ± 0.08) or downregulated (Hcn4, 0.16 ± 0.03 versus 0.37 ± 0.08; Kcna2, 0.35 ± 0.03 versus 0.80 ± 0.11; Kcna4, 0.79 ± 0.25 versus 1.90 ± 0.26; and Kcnj2, 0.52 ± 0.07 versus 0.78 ± 0.08) in GK ventricle compared with control ventricle. The amplitude of ventricular myocyte shortening and the intracellular Ca(2+) transient were unaltered; however, the time-to-peak shortening was prolonged and time-to-half decay of the Ca(2+) transient was shortened in GK myocytes compared with control myocytes. The results of this study demonstrate changes in expression of genes encoding various excitation-contraction coupling proteins that are associated with disturbances in myocyte shortening and intracellular Ca(2+) transport.

Troponin T and Plasma Collagen Peptides in Heart Failure

The extracellular matrix of the heart is made up of a number of structural proteins including fibrillar collagen, smaller amounts of elastin, laminin, fibronectin, and signaling peptides. The complex collagen 3-dimensional weave, mainly consisting of type I collagen, interconnects individual myocytes through a collagen–integrin–cytoskeletal–myofibril arrangement.1 This network supports cardiac myocytes during contraction and relaxation and also provides a mechanism for translating individual myocyte shortening and force generation into ventricular contraction. It is also responsible for much of the ventricle’s passive diastolic stiffness.2 In both human and animal studies, progressive left ventricular remodeling and dysfunction are associated with significant changes in the extracellular matrix.3–5 Article see p 406 The structural hallmark of prolonged pressure-overload hypertrophy is increased collagen accumulation between individual myocytes and myocyte fascicles (Figure 1).6,7 Thus, the highly organized architecture of the extracellular matrix undergoes significant alterations in collagen structure, composition, and geometry caused by increased collagen synthesis, postsynthetic processing, posttranslational modification, and decreased degradation and turnover. This “reactive” collagen deposition is characterized by both perivascular and interstitial fibrosis.2,8,9 The changes in collagen homeostasis that occur during the development of chronic pressure-overload hypertrophy are directly associated with increased myocardial diastolic stiffness properties, which in turn cause abnormal diastolic filling.7,10,11 Indeed, clinical evidence suggests that progressive extracellular matrix accumulation and diastolic dysfunction are important underlying pathophysiological mechanisms for heart failure in patients with pressure-overload hypertrophy.12,13 Figure 1. Scanning electron micrographs taken from normal nonhuman primate left ventricular myocardium following the induction of pressure-overload hypertrophy (POH). These microscopic studies demonstrated thickening of the collagen weave and overall increased relative content between myocytes with POH. Reproduced with permission.6 In volume-overload hypertrophy because of the persistently elevated preload, a much different pattern of extracellular …

Validation of an in vitro contractility assay using canine ventricular myocytes

Measurement of cardiac contractility is a logical part of pre-clinical safety assessment in a drug discovery project, particularly if a risk has been identified or is suspected based on the primary- or non-target pharmacology. However, there are limited validated assays available that can be used to screen several compounds in order to identify and eliminate inotropic liability from a chemical series. We have therefore sought to develop an in vitro model with sufficient throughput for this purpose.Dog ventricular myocytes were isolated using a collagenase perfusion technique and placed in a perfused recording chamber on the stage of a microscope at ~36°C. Myocytes were stimulated to contract at a pacing frequency of 1Hz and a digital, cell geometry measurement system (IonOptix™) was used to measure sarcomere shortening in single myocytes. After perfusion with vehicle (0.1% DMSO), concentration–effect curves were constructed for each compound in 4–30 myocytes taken from 1 or 2 dog hearts.The validation test-set was 22 negative and 8 positive inotropes, and 21 inactive compounds, as defined by their effect in dog, cynolomolgous monkey or humans. By comparing the outcome of the assay to the known in vivo contractility effects, the assay sensitivity was 81%, specificity was 75%, and accuracy was 78%.With a throughput of 6–8compounds/week from 1 cell isolation, this assay may be of value to drug discovery projects to screen for direct contractility effects and, if a hazard is identified, help identify inactive compounds.