Olivier Cazorla

Transmural stretch-dependent regulation of contractile properties in rat heart and its alteration after myocardial infarction

The “stretch‐sensitization” response is essential to the regulation of heart contractility. An increase in diastolic volume improves systolic contraction. The cellular mechanisms of this modulation, the Frank‐Starling law, are still uncertain. Moreover, their alterations in heart failure remains controversial. Here, using left ventricular skinned rat myocytes, we show a nonuniform stretch‐sensitization of myofilament activation across the ventricular wall. Stretch‐dependent Ca2+ sensitization of myofilaments increases from sub‐epicardium to sub‐endocardium and is correlated with an increase in passive tension. This passive tension‐dependent component of myofibrillar activation is not associated with expression of titin isoforms, changes in troponin I level, and phosphorylation status. Instead, we observe that stretch induces phosphorylation of ventricular myosin light chain 2 isoform (VLC2b) in sub‐endocardium specifically. Thus, VLC2b phosphorylation could act as a stretch‐dependent modulator of activation tuned within normal heart. Moreover, in postmyocardial infarcted rat, the gradient of stretch‐dependent Ca2+ sensitization disappears associated with a lack of VLC2b phosphorylation in sub‐endocardium. In conclusion, nonuniformity is a major characteristic of the normal adult left ventricle (LV). The heterogeneous myocardial deformation pattern might be caused not only by the morphological heterogeneity of the tissue in the LV wall, but also by the nonuniform contractile properties of the myocytes across the wall. The loss of a contractile transmural gradient after myocardial infarction should contribute to the impaired LV function.

Ca2+-Independent Alterations in Diastolic Sarcomere Length and Relaxation Kinetics in a Mouse Model of Lipotoxic Diabetic Cardiomyopathy

Previous studies demonstrated increased fatty acid uptake and metabolism in MHC-FATP transgenic mice that overexpress fatty acid transport protein (FATP)1 in the heart under the control of the &agr;-myosin heavy chain (&agr;-MHC) promoter. Doppler tissue imaging and hemodynamic measurements revealed diastolic dysfunction, in the absence of changes in systolic function. The experiments here directly test the hypothesis that the diastolic dysfunction in MHC-FATP mice reflects impaired ventricular myocyte contractile function. In vitro imaging of isolated adult MHC-FATP ventricular myocytes revealed that mean diastolic sarcomere length is significantly (P<0.01) shorter than in wild-type (WT) cells (1.79±0.01 versus 1.84±0.01 &mgr;m). In addition, the relaxation rate (dL/dt) is significantly (P<0.05) slower in MHC-FATP than WT myocytes (1.58±0.09 versus 1.92±0.13 &mgr;m/s), whereas both fractional shortening and contraction rates are not different. Application of 40 mmol/L 2,3-butadionemonoxime (a nonspecific ATPase inhibitor that relaxes actin–myosin interactions) increased diastolic sarcomere length in both WT and MHC-FATP myocytes to the same length, suggesting that MHC-FATP myocytes are partially activated at rest. Direct measurements of intracellular Ca2+ revealed that diastolic [Ca2+]i is unchanged in MHC-FATP myocytes and the rate of calcium removal is unexpectedly faster in MHC-FATP than WT myocytes. Moreover, diastolic sarcomere length in MHC-FATP and WT myocytes was unaffected by removal of extracellular Ca2+ or by buffering of intracellular Ca2+ with the Ca2+ chelator BAPTA (100 &mgr;mol/L), indicating that elevated intracellular Ca2+ does not underlie impaired diastolic function in MHC-FATP ventricular myocytes. Functional assessment of skinned myocytes, however, revealed that myofilament Ca2+ sensitivity is markedly increased in MHC-FATP, compared with WT, ventricular cells. In addition, biochemical experiments demonstrated increased expression of the &bgr;-MHC isoform in MHC-FATP, compared with WT ventricles, which likely contributes to the slower relaxation rate observed in MHC-FATP myocytes. Collectively, these data demonstrate that derangements in lipid metabolism in MHC-FATP ventricles, which are similar to those observed in the diabetic heart, result in impaired diastolic function that primarily reflects changes in myofilament function, rather than altered Ca2+ cycling.

SR33805, a Ca2+ antagonist with length-dependent Ca2+-sensitizing properties in cardiac myocytes

This study examined the effects of SR33805, a fantofarone derivative with reported strong Ca2+ ‐antagonistic properties, on the contractile properties of intact and skinned rat ventricular myocytes. On intact cells loaded with the Ca2+‐fluorescent indicator Indo‐1, the application of low concentrations of SR33805 enhanced the amplitude of unloaded cell shortening and decreased the duration of cell shortening. Amplitude of the Ca2+ transient was also decreased. These effects were accompanied with a shortening of the action potential and a dose‐dependent blockade of L‐type calcium current (IC50=2.4 × 10−8 M). On skinned cardiac cells, the application of a low SR33805 concentration (10−8 M) induced a significant increase in maximal Ca2+‐activated force at the two‐tested sarcomere lengths (SLs), 1.9 and 2.3 μm. The application of a larger dose of SR33805 (10−6–10−5 M) induced a significant leftward shift of the tension–pCa relation that accounts for Ca2+‐sensitization of the myofilaments, particularly at 2.3 μm SL. In conclusion, despite its strong Ca2+‐antagonistic properties SR33805 increases cardiac cell contractile activity as a consequence of its Ca2+‐sensitizing effects. These effects are attributable to both an increase in the maximal Ca2+‐activated force and a length‐dependent Ca2+‐sensitization.

Enhanced length-dependent Ca2+ activation in fish cardiomyocytes permits a large operating range of sarcomere lengths

Fish myocytes continue to develop active tension when stretched to sarcomere lengths (SLs) on the descending limb of the mammalian length-tension relationship. A greater length-dependent activation in fish than mammals could account for this because the increase in Ca(2+) sensitivity may overcome the tendency for force to fall due to reduced cross-bridge availability at SLs above optimal myofilament overlap. We stretched skinned fish and rat ventricular myocytes over a wide range of SLs, including those on the descending limb of the mammalian length-tension relationship. We found that fish myocytes developed greater active tension than rat myocytes at physiological Ca(2+) concentrations at long SLs as a result of a higher Ca(2+) sensitivity and a steeper relationship between Ca(2+) sensitivity and SL. We also investigated the diastolic properties of fish and rat myocytes at long SLs by measuring titin-based passive tension, titin isoform expression and titin phosphorylation. Fish myocytes produced higher titin-based passive tension despite expressing a higher proportion of a long N2BA-like isoform (38.0+/-2% of total vs 0% in rat). However, titin phosphorylation in fish myocytes was lower than in rat, which may explain some of the difference in passive tension between species. The high level of titin-based passive tension and the differential phosphorylation of sarcomeric proteins in fish myocytes may contribute to the enhanced length-dependent activation and underlie the extended range of in vivo stroke volumes found in fish compared with mammals.

Moderate exercise prevents impaired Ca2+ handling in heart of CO-exposed rat: implication for sensitivity to ischemia-reperfusion

Sustained urban carbon monoxide (CO) exposure exacerbates heart vulnerability to ischemia-reperfusion via deleterious effects on the antioxidant status and Ca(2+) homeostasis of cardiomyocytes. The aim of this work was to evaluate whether moderate exercise training prevents these effects. Wistar rats were randomly assigned to a control group and to CO groups, living during 4 wk in simulated urban CO pollution (30-100 parts/million, 12 h/day) with (CO-Ex) or sedentary without exercise (CO-Sed). The exercise procedure began 4 wk before CO exposure and was maintained twice a week in standard filtered air during CO exposure. On one set of rats, myocardial ischemia (30 min) and reperfusion (120 min) were performed on isolated perfused rat hearts. On another set of rats, myocardial antioxidant status and Ca(2+) handling were evaluated following environmental exposure. As a result, exercise training prevented CO-induced myocardial phenotypical changes. Indeed, exercise induced myocardial antioxidant status recovery in CO-exposed rats, which is accompanied by a normalization of sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a expression and then of Ca(2+) handling. Importantly, in CO-exposed rats, the normalization of cardiomyocyte phenotype with moderate exercise was associated with a restored sensitivity of the myocardium to ischemia-reperfusion. Indeed, CO-Ex rats presented a lower infarct size and a significant decrease of reperfusion arrhythmias compared with their sedentary counterparts. To conclude, moderate exercise, by preventing CO-induced Ca(2+) handling and myocardial antioxidant status alterations, reduces heart vulnerability to ischemia-reperfusion.

Carbon monoxide increases inducible NOS expression that mediates CO-induced myocardial damage during ischemia-reperfusion

We investigated the role of inducible nitric oxide (NO) synthase (iNOS) on ischemic myocardial damage in rats exposed to daily low nontoxic levels of carbon monoxide (CO). CO is a ubiquitous environmental pollutant that impacts on mortality and morbidity from cardiovascular diseases. We have previously shown that CO exposure aggravates myocardial ischemia-reperfusion (I/R) injury partly because of increased oxidative stress. Nevertheless, cellular mechanisms underlying cardiac CO toxicity remain hypothetical. Wistar rats were exposed to simulated urban CO pollution for 4 wk. First, the effects of CO exposure on NO production and NO synthase (NOS) expression were evaluated. Myocardial I/R was performed on isolated perfused hearts in the presence or absence of S-methyl-isothiourea (1 μM), a NOS inhibitor highly specific for iNOS. Finally, Ca(2+) handling was evaluated in isolated myocytes before and after an anoxia-reoxygenation performed with or without S-methyl-isothiourea or N-acetylcystein (20 μM), a nonspecific antioxidant. Our main results revealed that 1) CO exposure altered the pattern of NOS expression, which is characterized by increased neuronal NOS and iNOS expression; 2) cardiac NO production increased in CO rats because of its overexpression of iNOS; and 3) the use of a specific inhibitor of iNOS reduced myocardial hypersensitivity to I/R (infarct size, 29 vs. 51% of risk zone) in CO rat hearts. These last results are explained by the deleterious effects of NO and reactive oxygen species overproduction by iNOS on diastolic Ca(2+) overload and myofilaments Ca(2+) sensitivity. In conclusion, this study highlights the involvement of iNOS overexpression in the pathogenesis of simulated urban CO air pollution exposure.

Vascular endothelial function masks increased sympathetic vasopressor activity in rats with metabolic syndrome.

Sympathetic hyperactivation, a common feature of obesity and metabolic syndrome, is a key trigger of hypertension. However, some obese subjects with autonomic imbalance present a dissociation between sympathetic activity-mediated vasoconstriction and increased blood pressure. Here, we aimed to determine in a rat model of metabolic syndrome whether the endothelium endothelial nitric oxide (NO) synthase (eNOS)-NO pathway contributes to counteract the vasopressor effect of the sympathetic system. Rats were fed a high-fat and high-sucrose (HFS) diet for 15 wk. Sympathovagal balance was evaluated by spectral analysis of heart rate variability and plasmatic catecholamine measurements. Blood pressure was measured in the presence or absence of N-nitro-l-arginine methyl ester (l-NAME) to inhibit the contribution of eNOS. Vascular reactivity was assessed on isolated aortic rings in response to α1-adrenergic agonist. The HFS diet increased sympathetic tone, which is characterized by a higher low on the high-frequency spectral power ratio and a higher plasmatic concentration of epinephrine. Despite this, no change in blood pressure was observed. Interestingly, HFS rats exhibited vascular hyporeactivity (-23.6%) to α1-adrenergic receptor stimulation that was abolished by endothelial removal or eNOS inhibition (l-NAME). In addition, eNOS phosphorylation (Ser1177) was increased in response to phenylephrine in HFS rats only. Accordingly, eNOS inhibition in vivo revealed higher blood pressure in HFS rats compared with control rats (147 vs. 126 mmHg for mean blood pressure, respectively). Restrain of adrenergic vasopressor action by endothelium eNOS is increased in HFS rats and contributes to maintained blood pressure in the physiological range. NEW & NOTEWORTHY Despite the fact that prohypertensive sympathetic nervous system activity is markedly increased in rats with early metabolic syndrome, they present with normal blood pressure. These observations appear to be explained by increased endothelial nitric oxide synthase response to adrenergic stimulation, which results in vascular hyporeactivity to α-adrenergic stimulation, and therefore blood pressure is preserved in the physiological range. Listen to this articles corresponding podcast at http://www.physiology.org/doi/10.1152/ajpheart.00217.2017 .

0275 : Key role of eNOS in exercise-induced cardioprotection is dependent from coronary endothelium

To date, it is clearly accepted that the activation of endothelial isoform of NOS (eNOS) by exercise training constitutes a key trigger of exercise-induced cardioprotection against ischemia-reperfusion (IR). However, this enzyme is expressed both in coronary endothelial cells and cardiomyocytes, but the contribution of the one or the other one to such cardioprotection has never been challenged. The aim of this study was then to investigate the role of cardiomyocytes vs. endothelial eNOS in exercise cardioprotection. To this, rats were assigned to sedentary (Sed) or chronic aerobic exercised (Ex) group. Effects of exercise on vulnerability to IR or anoxia-reoxygenation (AR) were respectively evaluated at whole heart or cardiomyocytes and coronary artery levels. On a first set of rats, isolated cardiomyocytes were submitted to AR in presence or not of L-NAME, an eNOS inhibitor. Exercise reduced cells death and improved cells contractility after AR, but no effect of L-NAME was observed. Interestingly, exercise had no effect neither on eNOS phosphorylation on its activation site (Ser1177) nor on S-nitrosylation at cardiomyocytes level, whereas at whole heart level exercise increased both of them, suggesting that exercise impacted endothelial cells rather than cardiomyocytes. Then, to evaluate the contribution of endothelial cells on exercise-induced cardioprotection, on a Langendorff apparatus we treated hearts with Triton X-100 before IR to abolish coronary endothelial cells activity. Inactivation of endothelial cells totally suppressed cardioprotective effects of exercise. Finally, coronary arteries were isolated from hearts submitted to IR and endothelial function was assessed on a wire-myograph. We observed that alteration of endothelium-dependent coronary relaxation induced by IR was reduced in Ex group. In conclusion, these results show that coronary endothelial cells rather than cardiomyocytes play a key role in eNOS-dependent cardioprotection in Ex rat hearts.

0138 : Exercise training protects the heart against ischemia reperfusion in a mice model of diet-induced metabolic syndrome: no implication of the classic β3 adrenergic receptors – eNOS pathway

Metabolic syndrome is associated with a higher cardiac vulnerability to ischemia-reperfusion (IR). Regular exercise is recognized to protect the heart. This has been recently attributed to β3-adrenergic receptors (β3-AR) stimulation and subsequent increase in endothelial nitric oxide synthase (eNOS) activation. However, the role of this pathway in exercise-induced cardioprotection in animals with metabolic syndrome is unknown. We thus evaluated the role of the β3-AR/eNOS pathway in exercise-induced cardioprotection in a mouse model of metabolic syndrome. C57Bl6 mice were fed with high fat and sucrose diet (HFS) for 12 weeks and some had treadmill-exercise with a moderate intensity the last 4 weeks (HFS-Ex). First, HFS hearts were more sensitive to IR, which was prevented by exercise. Even though exercise protects the HFS heart, this was not associated with increased activation state of eNOS (level of eNOS-Pser1177 and the dimer/monomer ratio). Consequently, no increase in NO metabolites storage was observed after exercise in HFS hearts. This result may be explained by the loss of the β3-AR-eNOS pathway in HFS hearts. Indeed, the use of BRL37344, an agonist of β3-AR, increased eNOS-Pser1177 and protected the heart of Ctrl mice, whereas it had no effect in HFS hearts. Finally, considering that exercise-induced cardioprotection is also classically associated with increased antioxidant status, we next evaluated ROS production during early reperfusion and the subsequent activation of the apoptosis end-effector caspase 3. At early reperfusion we found that HFS increased ROS and caspase 3 activation. This phenomenon was blunted in HFS-Ex. Finally, a treatment with LPBNAH, a more amphiphilic nitrone antioxidant derived from PBN normalized HFS heart vulnerability to IR. To conclude these results showed that exercise-induced cardioprotection in HFS hearts is independent of the classical β3-AR-eNOS pathway, but involved oxidative stress during IR.

0034: Coronary endothelium plays a key role in exercise induced cardioprotection: a potential paracrine role of NO

We recently showed that endothelial isoform of NOS (eNOS) plays a key role in exercise-induced cardioprotection during myocardial ischemia reperfusion (IR). Although this enzyme is expressed in both coronary endothelial cells and cardiac myocytes, only 20% of the total cardiac eNOS seems to localize in the cardiomyocytes. The aim of this study was to investigate the role of coronary endothelium in the eNOS exercise-induced cardioprotection. Rats were assigned to sedentary (Sed) or exercised (Ex, 5 days/week for 5 weeks, 70% of maximal aerobic velocity) group. At the end of the exercise training period, hearts were mounted on an isolated hearts Langendorff apparatus and subjected to global ischemia (30 min) and reperfusion (120 minutes) in presence or not of a NOS inhibitor (L-NAME, 50xa0μM). Treatment with L-NAME of isolated hearts during IR abolished exercise-induced cardioprotection, confirming previous results on the role of eNOS during IR. The contribution of the coronary endothelial cells was prevented by perfusing a bolus injection of Triton X-100 in the coronary system before IR. When endothelial cells are inactivated, the beneficial effects of exercise on hearts sensitivity to IR were totally abolished. Moreover cardiomyocytes isolated from sedentary and exercised hearts were subjected to 1 hour anoxia and 1 hour reoxygenation in presence or not of L-NAME (50xa0μM). Interestingly, at the cardiomyocyte level, exercise-induced protection is not clear and L-NAME had no effect, suggesting that eNOS was not involved in such mechanism. In conclusion, the present results show that NO synthesis from coronary endothelial cells plays a major paracrine role in exercise induced cardioprotection.