Changes In Contractile Parameters Research Articles

Increased Myocardial Oxygen Consumption Precedes Contractile Dysfunction in Hypertrophic Cardiomyopathy Caused by Pathogenic TNNT2 Gene Variants

BackgroundHypertrophic cardiomyopathy is caused by pathogenic sarcomere gene variants. Individuals with a thin‐filament variant present with milder hypertrophy than carriers of thick‐filament variants, although prognosis is poorer. Herein, we defined if decreased energetic status of the heart is an early pathomechanism in TNNT2 (troponin T gene) variant carriers.Methods and ResultsFourteen individuals with TNNT2 variants (genotype positive), without left ventricular hypertrophy (G+/LVH−; n=6) and with LVH (G+/LVH+; n=8) and 14 healthy controls were included. All participants underwent cardiac magnetic resonance and [11C]‐acetate positron emission tomography imaging to assess LV myocardial oxygen consumption, contractile parameters and myocardial external efficiency. Cardiac efficiency was significantly reduced compared with controls in G+/LVH− and G+/LVH+. Lower myocardial external efficiency in G+/LVH− is explained by higher global and regional oxygen consumption compared with controls without changes in contractile parameters. Reduced myocardial external efficiency in G+/LVH+ is explained by the increase in LV mass and higher oxygen consumption. Septal oxygen consumption was significantly lower in G+/LVH+ compared with G+/LVH−. Although LV ejection fraction was higher in G+/LVH+, both systolic and diastolic strain parameters were lower compared with controls, which was most evident in the hypertrophied septal wall.ConclusionsUsing cardiac magnetic resonance and [11C]‐acetate positron emission tomography imaging, we show that G+/LVH− have an initial increase in oxygen consumption preceding contractile dysfunction and cardiac hypertrophy, followed by a decline in oxygen consumption in G+/LVH+. This suggests that high oxygen consumption and reduced myocardial external efficiency characterize the early gene variant–mediated disease mechanisms that may be used for early diagnosis and development of preventive treatments.

Chronic drug-induced effects on contractile motion properties and cardiac biomarkers in human induced pluripotent stem cell-derived cardiomyocytes.

In the pharmaceutical industry risk assessments of chronic cardiac safety liabilities are mostly performed during late stages of preclinical drug development using in vivo animal models. Here, we explored the potential of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) to detect chronic cardiac risks such as drug-induced cardiomyocyte toxicity. Video microscopy-based motion field imaging was applied to evaluate the chronic effect (over 72h) of cardiotoxic drugs on the contractile motion of hiPS-CMs. In parallel, the release of cardiac troponin I (cTnI), heart fatty acid binding protein (FABP3) and N-terminal pro-brain natriuretic peptide (NT-proBNP) was analysed from cell medium, and transcriptional profiling of hiPS-CMs was done at the end of the experiment. Different cardiotoxic drugs altered the contractile motion properties of hiPS-CMs together with increasing the release of cardiac biomarkers. FABP3 and cTnI were shown to be potential surrogates to predict cardiotoxicity in hiPS-CMs, whereas NT-proBNP seemed to be a less valuable biomarker. Furthermore, drug-induced cardiotoxicity produced by chronic exposure of hiPS-CMs to arsenic trioxide, doxorubicin or panobinostat was associated with different profiles of changes in contractile parameters, biomarker release and transcriptional expression. We have shown that a parallel assessment of motion field imaging-derived contractile properties, release of biomarkers and transcriptional changes can detect diverse mechanisms of chronic drug-induced cardiac liabilities in hiPS-CMs. Hence, hiPS-CMs could potentially improve and accelerate cardiovascular de-risking of compounds at earlier stages of drug discovery. This article is part of a themed section on New Insights into Cardiotoxicity Caused by Chemotherapeutic Agents. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.21/issuetoc.

Paracrine Effects of IGF-1 Overexpression on the Functional Decline Due to Skeletal Muscle Disuse: Molecular and Functional Evaluation in Hindlimb Unloaded MLC/mIgf-1 Transgenic Mice

Slow-twitch muscles, devoted to postural maintenance, experience atrophy and weakness during muscle disuse due to bed-rest, aging or spaceflight. These conditions impair motion activities and can have survival implications. Human and animal studies demonstrate the anabolic role of IGF-1 on skeletal muscle suggesting its interest as a muscle disuse countermeasure. Thus, we tested the role of IGF-1 overexpression on skeletal muscle alteration due to hindlimb unloading (HU) by using MLC/mIgf-1 transgenic mice expressing IGF-1 under the transcriptional control of MLC promoter, selectively activated in skeletal muscle. HU produced atrophy in soleus muscle, in terms of muscle weight and fiber cross-sectional area (CSA) reduction, and up-regulation of atrophy gene MuRF1. In parallel, the disuse-induced slow-to-fast fiber transition was confirmed by an increase of the fast-type of the Myosin Heavy Chain (MHC), a decrease of PGC-1α expression and an increase of histone deacetylase-5 (HDAC5). Consistently, functional parameters such as the resting chloride conductance (gCl) together with ClC-1 chloride channel expression were increased and the contractile parameters were modified in soleus muscle of HU mice. Surprisingly, IGF-1 overexpression in HU mice was unable to counteract the loss of muscle weight and the decrease of fiber CSA. However, the expression of MuRF1 was recovered, suggesting early effects on muscle atrophy. Although the expression of PGC-1α and MHC were not improved in IGF-1-HU mice, the expression of HDAC5 was recovered. Importantly, the HU-induced increase of gCl was fully contrasted in IGF-1 transgenic mice, as well as the changes in contractile parameters. These results indicate that, even if local expression does not seem to attenuate HU-induced atrophy and slow-to-fast phenotype transition, it exerts early molecular effects on gene expression which can counteract the HU-induced modification of electrical and contractile properties. MuRF1 and HDAC5 can be attractive therapeutic targets for pharmacological countermeasures and then deserve further investigations.

Mechanism and significance of early, rapid shortening in sensitized airway smooth muscleThis article is one of a selection of papers published in the Special Issue on Recent Advances in Asthma Research.

It has been reported that sensitization of animals to allergens increases both early shortening velocity and myosin light-chain kinase of their airway smooth muscle without increasing force generated by these muscles. Since early shortening sets muscle length for the duration of a contraction, these responses might be expected to produce greater airway obstruction. Here, it is explained how the more rapid early shortening without increased force production is predicted by the 2-stage process of activation followed by contraction posited by the crossbridge theory of contraction when the rate, but not the extent, of activation is increased. The experimental results are reproduced by a simple model in which activation rate is increased 1.6-fold without any other changes in contractile parameters. These results reinforce suggestions that sensitized animals are a model for reactive airway disease.

Filament lattice changes in smooth muscle assessed using birefringence

The long functional range of some types of smooth muscle has been the subject of recent study. It has been proposed that the muscle filament lattice adapts to longer lengths by placing more filaments in series and that lattice plasticity is facilitated by myosin filament evanescence, with filaments dissociating during relaxation and reforming upon activation. Support for these dynamic changes in the filament lattice has been provided partly by changes in contractile parameters at different times in the contraction-relaxation cycle at different lengths. If the changes in contractile parameters result from filament formation and dissociation, these structural changes must occur on the time scale of tension development and relaxation. To assess whether thick-filament formation could account for the contractile changes, we measured birefringence continuously during activation and relaxation and compared these optical changes with the time course of force development and relaxation. Birefringence is a well-known property of muscle; striations in skeletal and cardiac muscle result from the A-bands being anisotropic, i.e., birefringent, and it is now known that this optical property is due to the presence of myosin thick filaments in the A-bands. Thus, the strength of birefringence is expected to represent the density of thick filaments. Here, we describe the principle of the method, the techniques for recording the optical signals, some initial results, and discuss the interpretation of results and some limitations of the method.

Effects of chronic endothelin-1 stimulation on cardiac myocyte contractile function.

Endothelin-1 (ET-1) has acute positive inotropic effects, but consequences of chronically increased ET-1 on contractile function of cardiac myocytes are largely unknown. In the present study, effects of long-term treatment with ET-1 (10 nM) for 5 days on both force development [force of contraction (FOC)] and kinetics of contraction were determined in heart tissue reconstituted from rat cardiac cells. Isometric force was measured in response to cumulative concentrations of Ca(2+) and isoprenaline. ET-1 augmented basal FOC by 64 +/- 11% (P < 0.05), which was associated with a significantly blunted contractile response to Ca(2+) and isoprenaline. Moreover, ET-1 significantly prolonged relaxation (62 +/- 3 vs. 53 +/- 2 ms). Selective ET(A) (BQ-123) and ET(B) receptor blockade (BQ-788) demonstrated that effects of ET-1 on contractile function were mediated through the ET(A) receptor subtype. Effects of ET-1 were prevented by cotreatment with either Ro31-8425, a PKC inhibitor, or dimethylamiloride, an inhibitor of the Na(+)/H(+) exchanger. In contrast to long-term ET-1 treatment, no changes in contractile parameters were observed after ET-1 treatment for 3 h before force measurement. These data suggest that chronic ET-1 stimulation has dual effects on contractility: improvement of basal force but impairment of twitch kinetics and inotropic responsiveness to beta-adrenoceptor stimulation. The signaling pathways involved include ET(A) receptors, PKC, and the Na(+)/H(+) exchanger. The present in vitro findings raise the possibility that ET-1 may exert both adaptive and maladaptive effects in the failing myocardium in which local accumulation of ET-1 is present.

Cross-influence of membrane polyunsaturated fatty acids and hypoxia-reoxygenation on alpha- and beta-adrenergic function of rat cardiomyocytes.

The purpose of the present investigation was to determine whether the beneficial effects of polyunsaturated fatty acids (PUFA) may influence ischemia-reperfusion-induced alterations of myocardial alpha- and beta-adrenoceptor (alpha-AR, beta-AR) responsiveness. This study was carried out using monolayer cultures of neonatal rat ventricular myocytes in a substrate-free, hypoxia-reoxygenation model of ischemia. The cardiomyocytes (CM) were incubated during 4 days in media enriched either with n-6 PUFA (arachidonic acid, AA) or with n-3 PUFA (eicosapentaenoic acid, EPA, and docosahexaenoic acid, DHA). The n-6/n-3 ratio in n-3 CM was close to 1.2, compared to 20.1 in n-6 CM. The contractile parameters of n-6 CM and n-3 CM were similar in basal conditions as well as during hypoxia and reoxygenation. In basal conditions, the phospholipid (PL) enrichment with long chain n-3 PUFA resulted in an increased chronotropic response to isoproterenol (ISO) and to phenylephrine (PHE). After posthypoxic reoxygenation, the chronotropic response to beta-AR activation in n-6 CM was significantly enhanced as compared with the control response in normoxia. In opposition, the ISO-induced rise in frequency in n-3 CM in control normoxia and after reoxygenation was similar. In these n-3 CM, the changes in contractile parameters, which accompanied the chronotropic response, were also similar in reoxygenation and in normoxic periods, although the rise in shortening velocity was slightly increased after reoxygenation. In response to PHE addition, only the chronotropic effect of n-6 CM appeared significantly enhanced after hypoxic treatment. These results suggested that increasing n-3 PUFA in PL reduced the increase in alpha- and beta-AR functional responses observed after hypoxia-reoxygenation. This effect may partly account for the assumed cardiac protective effect of n-3 PUFA, through the attenuation of the functional response to catecholamines in the ischemic myocardium.

Effect of ablation of phospholamban on dynamics of cardiac myocyte contraction and intracellular Ca2+.

We compared mechanical activity and Ca2+ transients of ventricular myocytes isolated from wild-type and phospholamban (PLB)-deficient mouse hearts in control conditions and during beta-adrenergic stimulation. Compared with wild-type controls, cells isolated from PLB-deficient mouse hearts showed 1) a 2-fold increase in extent of cell shortening, 2) a 3-fold increase in maximal shortening velocity, and 3) a 3.4-fold increase in maximal relengthening velocity. PLB-deficient myocytes also demonstrated significant increases in the peak amplitude of the fura 2 fluorescence ratio and the rates of rising and falling phases of the Ca2+ transient. The fura 2 diastolic ratios were similar in both groups, suggesting no change in intracellular Ca2+ during diastole. In PLB-deficient myocytes, 0.05 microM isoproterenol induced an increase in the twitch amplitude by 152 +/- 11% (n = 6) compared with 290 +/- 31% (n = 6) in wild-type cells. Maximal shortening velocity was increased by 183 +/- 10% (n = 6) in PLB-deficient myocytes, compared with 398 +/- 62% (n = 6) in wild-type cells. The isoproterenol-induced increase in maximum relengthening velocity was increased by 168 +/- 8% (n = 6) in PLB-deficient cells compared with 445 +/- 71% (n = 6) in wild-type myocytes. In both groups, these changes in contractile parameters were accompanied by changes in the Ca2+ transient. Our results indicate that phosphorylation of sites other than PLB may play an important role in regulation of contraction-relaxation dynamics of heart cells responding to beta-adrenergic stimulation.

Effects of carbonic anhydrase inhibitors on oxygen consumption and lactate accumulation in skeletal muscle

In isolated rat soleus and extensor digitorum longus (EDL) muscles, the effects of carbonic anhydrase inhibitors were studied on oxygen consumption as well as lactate release and accumulation after incubation in inhibitors lasting long enough to produce marked changes in contractile parameters and in the concentrations of energy-rich phosphates. The inhibitors used were chlorzolamide (10(-3) M) and NaCNO (10(-2) M). Compared with control muscles, muscles treated with either of the two inhibitors showed a decrease in force, and an increase in time-to-peak as well as in relaxation time. Lactate content and release in soleus and in EDL were increased by factors of 2-3 with both inhibitors. With both inhibitors, oxygen consumption in the red soleus increased by approximately 27%, whereas in EDL, no significant change could be observed. The increase in aerobic metabolic rate in the red soleus only might indicate that the isozyme CA III, which is present only in this type of muscle, is in some way involved in keeping the oxygen consumption low. The increase in anaerobic metabolic rate occurring in both muscles can possibly be explained by increases in Pi and ADP.

Differential sensitivity to isoprenaline of troponin I and phospholamban phosphorylation in isolated rat hearts

Phosphorylation of phospholamban (PLB), a membrane-bound 15 kDa protein and troponin I (TNI) was studied in isolated perfused rat hearts by using the back-phosphorylation technique with [32P]ATP catalysed by an excess of exogenous catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase, followed by protein separation. This standardized method allows the quantitative detection of protein phosphorylation specifically stimulated by cAMP. In control hearts the extent of specific phosphorylation was equivalent to 3.3 nmol of PLB and 11.0 mumol of TNI per g of cardiac tissue. In hearts freeze-clamped 30 s after exposure to isoprenaline (10 pM-10 microM), there was a dose-dependent decrease in phosphate incorporation in vitro, indicating a phosphorylation of the respective proteins in vivo. A differential sensitivity of TNI and PLB phosphorylation towards the beta-adrenergic agonist and the subsequent increase in tissue cAMP was found, favouring TNI phosphorylation. K0.5 values for isoprenaline were 2.94 +/- 0.04 nM and 4.46 +/- 0.24 nM for PLB and the 15 kDa protein, but 0.13 +/- 0.01 nM for TNI phosphorylation in the intact tissue. At an isoprenaline-induced increase in cAMP less than 3 pmol/mg of protein there was no or only a small increase in PLB phosphorylation, whereas TNI phosphorylation was nearly maximal. By plotting phosphorylation data against changes in contractile parameters a strong correlation was obtained for TNI (r = 0.95), assuming a linear relationship. For PLB a complex relationship is likely to exist. Our data (i) indicate a functional compartmentalization of the cAMP signal cascade and (ii) confirm that phosphorylation of TNI rather than of PLB is related to changes in mechanical myocardial responses.

Time course of structure, function, and metabolic changes due to an exogenous source of oxygen metabolites in rat heart

Effects of xanthine (2 mM) and xanthine oxidase (10 U/L) perfusion on myocardial function, lipid peroxide content, high-energy phosphates and their metabolites, and ultrastructure were examined in isolated perfused rat hearts to define the time course of myocardial injury due to exogenous supply of active oxygen species. Peak-developed force and dF/dt showed a decline within 5 min and complete contractile failure was seen at 20 min. Resting tension was higher at 10 min and reached a maximum value of 400% at 40 min. These changes in contractile parameters were reduced by superoxide dismutase (1.2 x 10(5) U/L), catalase (2 and 4 X 10(4) U/L), and mannitol (10 and 20 mM). Lipid peroxide content was significantly higher at 5 min and rose continuously with xanthine-xanthine oxidase (X-XO) perfusion. A close correlation was noted (r = 0.935) between increased lipid peroxide content and a decrease in peak-developed force. Creatine phosphate and adenosine triphosphate (ATP) showed a time-dependent decrease due to X-XO perfusion. Loss of ATP also correlated (r = 0.819) with the contractile failure. Adenosine diphosphate showed an increase at 5 min followed by a decrease at 20 and 40 min. Adenosine monophosphate, adenosine, and creatine content increased with X-XO perfusion. In a semiquantitative morphometric study, significant myocardial and vascular changes became apparent only after 10 min of X-XO perfusion. When a 5-min perfusion with X-XO was followed by a control perfusion, a recovery of developed force and normal structure was noted at 40 min. These data show that X-XO induced contractile failure involves partially reduced forms of oxygen such as superoxide, hydroxyl radicals, and hydrogen peroxide. The negative inotropic effect of a vascular supply of these active oxygen species may be related to increased lipid peroxidation as well as the loss of high-energy phosphates. Structural damage to myocytes and blood vessels and a rise in resting tension were delayed events requiring a continuous and longer exposure to radical species.

Carbonic anhydrase inhibition affects contraction of directly stimulated rat soleus

We have studied the contractile parameters of directly stimulated isolated rat soleus muscles incubated in media containing the carbonic anhydrase inhibitors chlorzolamide (5·10 −4M) or cyanate (10 −2M). Both inhibitors caused a decrease in isometric twitch and tetanic (5s) tensions and an increase in muscle relaxation time. It is speculated that among the three types of skeletal muscle carbonic anhydrase it may be the enzyme associated with the sarcoplasmatic reticulum whose inhibition caused the observed changes in contractile parameters.