PharmD Pierre-Edouard Grillet

Quentin Wynands Msc

MD Fares Gouzi

Patrice Bideaux

Aurelie Fort Phd

Valérie Scheuermann Msc

PhD Alain Lacampagne

PhD Anne Virsolvy

PhD Jérôme Thireau

PhD Pieter De Tombe

MD Arnaud Bourdin

PhyMedExp Olivier Cazorla
email: olivier.cazorla@inserm.fr

Diastolic cardiomyopathy secondary to experimentally induced exacerbated emphysema

Keywords: elastase, cardiac myofilaments, calcium, COPD, HFpEF AMP: adenosine mono-phosphate COPD: chronic obstructive pulmonary disease ECG: electrocardiogram HFpEF: heart failure with preserved ejection fraction LVEF: left ventricular ejection fraction LPS: lipopolysaccharide PKA: protein kinase A SL: sarcomere length TAPSE: tricuspid annular plane systolic excursion TDI: tissue Doppler imaging

Chronic Obstructive Pulmonary Disease (COPD) is a clinical entity of increasing significance. COPD involves abnormalities of the airways and in emphysema, parenchymal pulmonary destruction. Cardiovascular disease has emerged as a significant comorbidity to COPD. Heart Failure with preserved Ejection Fraction (HFpEF) appears to be particularly associated with COPD-Emphysema. Traditional treatments have shown limited efficacy in improving COPD associated HFpEF. This lack of therapeutic efficacy highlights the need to identify potential mechanisms that link COPD-Emphysema to HFpEF. Therefore, we aimed to study the delayed cardiac physiological impacts in a rat model with acute exacerbated emphysema. Emphysema was induced by 4 weekly 4UI elastase intra-tracheal pulmonary instillations and exacerbation by one final additional LPS instillation in male Wistar rats. At 5 weeks following the LPS/elastase exposure, in-vivo and ex-vivo pulmonary and cardiac measurements were performed. Experimental exacerbated emphysema resulted in decreased pulmonary function and exercise intolerance. Histological analysis revealed parenchymal pulmonary destruction without signs of inflammation or cardiac fibrosis. In-vivo cardiac functional analysis revealed diastolic dysfunction and tachycardia. Ex-vivo analysis revealed a cellular cardiomyopathy with decreased myofilament Ca 2+ sensitivity, cross-bridge cycling kinetics and increased adrenergic PKA-dependent phosphorylation of troponin-I. Experimental exacerbated emphysema was associated with exercise intolerance that appeared to be secondary increased β-adrenergic tone and subsequent cardiac myofilament dysfunction. A β1-receptor antagonist treatment (bisoprolol) started 24h post ELA-LPS instillation prevented in-vivo and ex-vivo diastolic dysfunction. These results suggest that novel treatment strategies targeted to the cardiac myofilament may be beneficial to combat exacerbated emphysema associated HFpEF.

Translational perspectives

• Exacerbated emphysema animal model is suitable to mimic the physiopathology of COPD patients, especially cardiac alterations consisting in diastolic heart failure with preserved ejection fraction.

•

Ex-vivo analysis revealed a cellular cardiomyopathy probably caused by increased circulating catecholamines and chronotropic incompetence. • β1-blocker therapy appears to be relevant to reduce diastolic dysfunction, chronotropic incompetence and cellular changes.

•

Further investigation is warranted to validate the mechanism by which selective β1 receptor antagonism reduce or delay the risk of cardiomyopathy in COPD patients.

INTRODUCTION

Chronic Obstructive Pulmonary Disease (COPD) is a clinical entity of increasing significance towards morbidity and mortality worldwide 1,2 . COPD involves abnormalities of the airways and, in emphysema, parenchymal pulmonary destruction. Emphysema may lead to pulmonary hyperinflation as a consequence of blunted lung elastic recoil and gas trapping of pulmonary air 1 . In addition, cardiovascular disease has emerged as a significant comorbidity to COPD, irrespective of shared risk factors such as cigarette smoking, pollution, sedentary lifestyle and ageing 3 . Moreover, large cohort studies support the notion of a crosstalk between COPD-Emphysema and cardiovascular disease 4,5 .

Diastolic heart failure (HF), also termed heart failure with preserved ejection fraction (HFpEF), appears to be particularly associated with severe COPD-Emphysema 4,6 . Conversely, prognosis in HFpEF is worsened in the presence of COPD 5 . Treatments such as inhaled corticosteroids, statins, and diuretics have shown limited efficacy in improving COPD associated HFpEF 7 , consistent with the observation that traditional therapeutic strategies aimed to combat systolic heart failure have also been largely unsuccessful to treat HFpEF 8 . Diastolic dysfunction, the hallmark of HFpEF, has been attributed to three main cellular mechanisms: cardiac fibrosis with increased tissue stiffness, deranged calcium homeostasis, and post-translational modifications of myofilament's proteins 9 . However, the contribution of COPD in these pathways is yet to be determined. Consequently, the lack of therapeutic efficacy to ameliorate HFpEF, particularly in COPD patients, highlights the need to identify potential cellular mechanisms that link COPD-Emphysema to HFpEF 10,[START_REF] Heidenreich | AHA/ACC/HFSA Guideline for the Management of Heart Failure[END_REF] .

Few studies have examined mechanisms that may underlie development of HFpEF in COPD-Emphysema patients. One possibility is the impact of pulmonary hyperinflation on cardiac preload via a mechanical constraint imposed onto the heart. Indeed, surgically [START_REF] Jörgensen | Effects of Lung Volume Reduction Surgery on Left Ventricular Diastolic Filling and Dimensions in Patients with Severe Emphysema[END_REF] or pharmacologically [START_REF] Hohlfeld | Effect of lung deflation with indacaterol plus glycopyrronium on ventricular filling in patients with hyperinflation and COPD (CLAIM): a double-blind, randomised, crossover, placebo-controlled, single-centre trial[END_REF] induced lung deflation has been shown to improve cardiac function. In addition, increased pulmonary vascular resistance may cause yet another extra-cardiac limitation to cardiac function in COPD [START_REF] Rabe | Cardiovascular disease and COPD: dangerous liaisons?[END_REF] . However, intra-cardiac causes of cardiac dysfunction in COPD-Emphysema cannot be excluded, but are yet to be examined.

METHODS

An expanded Methods section is available in the on-line supplement.

Animal Model

All investigations conformed to European Parliament Directive 2010/63/EU and were approved by the local ethics committee (APAFIS#13133-2018012212031585v2). Experimental emphysema was induced in seven-weekold sedated male rats (1% isoflurane) by intra-tracheal instillation of pancreatic porcine elastase (ELA, 4 units/200µL saline solution, Sigma-Aldrich, France) once a week for four weeks (Fig. 1A). To mimic COPD exacerbations related COPD patients display increased vulnerability to pulmonary virus and/or bacterial infections that can induce acute exacerbations of the disease and systemic inflammation [START_REF] Oudijk | Systemic inflammation in chronic obstructive pulmonary disease[END_REF] . In the context of COPD, the delayed impact of transient pulmonary inflammation in presence of emphysema on cardiac function has not been examined in detail.

Animal models reproducing exacerbated emphysema associated with cardiac dysfunction are essential to determine cellular and molecular mechanisms underlying the syndrome. Since the 1970s several experimental models have used single-dose [START_REF] Buczek-Thomas | Elastase Mediates the Release of Growth Factors from Lung In Vivo[END_REF] treatment or more recently multiple-dose elastase treatment to induce emphysema. Among these, some studies have explored cardiac alterations, especially on the right side and pulmonary artery. Indeed, right heart dilatation and hypertrophy correlated to emphysema [START_REF] Petta | Effects of the association of diabetes and pulmonary emphysema on cardiac structure and function in rats[END_REF] , pulmonary artery remodeling and hypoxic hyperactivity [START_REF] Sato | Pulmonary haemodynamics in awake rats following treatment with endotracheal pancreatic elastase[END_REF] , and echocardiographic documented alterations of pulmonary artery function (PAT/PET) [START_REF] Padilha G De | Comparison between effects of pressure support and pressure-controlled ventilation on lung and diaphragmatic damage in experimental emphysema[END_REF] have been described in elastaseinduced emphysema rat models. Lately these experimental models have been combined with lipopolysaccharide (LPS) treatment to mimic acute pulmonary infection/exacerbation. Accordingly, in the present study we adapted these mouse [START_REF] Ceelen | Altered protein turnover signaling and myogenesis during impaired recovery of inflammation-induced muscle atrophy in emphysematous mice[END_REF] and rat 21 models to study the delayed impact of transient pulmonary inflammation in presence of emphysema, on cardiac function. We used a multi-dose treatment exacerbated emphysema model, that is based on four weekly intra-tracheal pulmonary instillations of elastase, followed by a final elastase instillation combined with LPS. A significant finding of our study is exacerbated emphysema induced diastolic heart failure that may be the result of molecular cellular alterations of cardiac myocytes, and in particular the cardiac myofilament.

to recurrent pulmonary infections, rats received with the last ELA instillation, a single dose of LPS (E.coli O55:B5, 2.5mg/kg, Sigma-Aldrich, France). Control animals were handled identically but did not receive any instillation. Next, animals were kept for 5 weeks following the last pulmonary instillation to allow for examination of the functional impacts of exacerbation-emphysema that remained after the acute inflammatory phase post ELA-LPS. In addition, a subgroup of animals was treated with a  1 -receptor antagonist (2.5mg/kg/day in drinking water, bisoprolol hemifumarate, MedChemExpress, USA) starting 24h post ELA-LPS instillation (Fig. 1A). Rats were euthanized with sodium pentobarbital overdose (140mg/kg, i.p.).

In-vivo respiratory and cardiac functions and exercise tolerance

In-vivo physiological functions were tested one day before sacrifice including:

Respiratory function using a whole-body plethysmograph in awake animals as previously described [START_REF] Amancio G De Cs | Effect of pyridostigmine on in vivo and in vitro respiratory muscle of mdx mice[END_REF] . Parameters included respiratory rate, minute volume, peak inspiratory flow, and peak expiratory flow.

Maximum Oxygen consumption, assessed by an incremental exercise test on a metabolic treadmill, as well as VO 2max and maximum running distance.

Cardiovascular function, explored by echocardiography on anesthetized animals (Isoflurane, 2.5-3.5%) as previously described [START_REF] Chakouri | Stress-induced protein S-glutathionylation and phosphorylation crosstalk in cardiac sarcomeric proteins -Impact on heart function[END_REF] and non-invasive systemic pressure by tail-cuff after familiarization of animals to the procedure. Electrocardiograms (ECG), using telemetric transmitter for 24h during normal activity in the cage as previously described [START_REF] Andre | Carbon monoxide pollution promotes cardiac remodeling and ventricular arrhythmia in healthy rats[END_REF] . For dobutamine testing (2mg/kg, i.p), ECGs were recorded 4 hours pre-and 4 hours post-injection.

Histological characterization of emphysema, fibrosis and inflammation

For histological analysis, explanted lungs were inflated to 25cm H 2 O pressure with 4% formol by means of tracheal cannulation, and then immersed in 4%-formol. Mean linear intercept was measured on lung tissue sections as previously described [START_REF] Thurlbeck | Internal surface area and other measurements in emphysema[END_REF] . The heart was either directly immersed in 4%-formol for histology, or rapidly frozen with liquid N 2 cooled clamps followed by -80°C storage for biochemistry or myofilament studies. Collagen content was evaluated on cardiac tissue sections using picrosirius red staining.

Western-Blot analysis

7

RESULTS

Exacerbated emphysema rat model

The experimental protocol employed to induce exacerbated emphysema is schematically illustrated in Figure 1A. Five weeks following elastase and LPS tracheal instillations, post-mortem lung tissue histological analysis Cyclic AMP dependent protein kinase (PKA) dependent myocardial protein phosphorylation was determined by Western-Blot analysis as previously described [START_REF] Andre | Subendocardial increase in reactive oxygen species production affects regional contractile function in ischemic heart failure[END_REF] . Frozen LV tissue was pulverized on dry-ice, solubilized in nonreducing Laemmli buffer followed by SDS-PAGE and Western-Blot. Phosphorylation levels were expressed relative to total protein content.

Single cardiomyocyte analysis

Membrane intact cardiomyocytes were isolated from the heart by enzymatic digestion as previously described [START_REF] Andre | Carbon monoxide pollution promotes cardiac remodeling and ventricular arrhythmia in healthy rats[END_REF] . To analyze cell shortening and Ca 2+ homeostasis, signals of sarcomere length (SL) and Indo-1 ratiometric fluorescent Ca 2+ indicator were recorded at 1 Hz (IonOptix system).

Cardiac myofilament function was assessed in permeabilized cardiomyocytes obtained from the frozen samples as previously described [START_REF] Cazorla | Transmural stretch-dependent regulation of contractile properties in rat heart and its alteration after myocardial infarction[END_REF] . Parameters calculated included maximum Ca 2+ induced myofilament force and Ca 2+ sensitivity.

Statistical analysis

Data are presented as mean±SEM or as median with total distribution and 25 -50% interquartile. Differences were assessed either by the Student's t-test or by the Mann Whitney's rank test whenever the data distribution was found not to be Gaussian as determined by the Shapiro-Wilk's normality test (GraphPad Prism). Analysis of differences for three groups was assessed by one-way ANOVA analysis followed by Bonferonni's multiplecomparisons test or Kruskal-Wallis test with Dunn's correction for the multiple comparison post hoc test. p<0.05 was considered significant. revealed significant parenchymal alveolar destruction as presented in Figure 1B. The resulting alveolar enlargement, as quantified by the mean linear intercept dimension parameter, was increased by ~50% in the emphysema group. In addition, we found no evidence of ELA-LPS induced major chronic inflammation measured by pulmonary immunohistochemistry or systemic plasma cytokine and chemokine analysis (cf. Supplemental Figure 1).

In-vivo whole-body plethysmography confirmed the development of exacerbated emphysema as illustrated in Figure 1. In particular, peak expiratory flow was significantly reduced in the ELA-LPS group (Fig. 1D). Peak inspiratory flow, in contrast, was not impacted by ELA-LPS treatment (Fig. 1C), as were other parameters such as respiratory rate and minute volume (cf. Supplemental Table 1).

Maximal oxygen uptake (VO 2max ), as measured during an incremental metabolic treadmill exercise test, was significantly reduced in the ELA-LPS group (Fig. 1E), as was the maximum running distance (Fig. 1F).

Overall, these in-vivo functional physiological data are consistent with presence of exacerbated emphysema and a concomitant reduction in global exercise capacity.

Functional signs of HFpEF

At the conclusion of the five-week incubation period, ELA-LPS animals displayed a ~20% increase in heart rate as measured by ECG telemetry (cf. Supplemental Table 2). Mean arterial pressure assessed by the tail-cuff method was also significantly increased in ELA-LPS animals (119.9±2.4 mmHg, n=15) compared to control animals (107.2±1.8 mmHg, n=11). Echocardiographic evaluation was next performed in lightly sedated (isoflurane) animals.

Since heart rate is a confounding factor in echocardiography, the isoflurane dosage was adjusted to achieve similar heart rates (~350 bpm) in control and ELA-LPS animals. Echocardiographic assessed parameters included M-mode chamber dimensions (Fig. 2A), mitral-flow Doppler (Fig. 2B), and mitral-tissue Doppler (Fig. 2C). A complete summary of the acquired measurements can be found in the supplementary data section (cf. Supplemental Table 3).

ELA-LPS treatment resulted in unchanged left ventricular ejection fraction (LVEF%; Fig. 2A), but increased E/A (Fig. 2B) and E/e' (Fig. 2C) ratios, as well as a ~10% reduction in cardiac output (cf. supplement Table 3). There was no apparent echocardiographic alteration in right ventricular systolic function as indexed by TAPSE and S tricuspid wave velocity (Supplemental Table 3). These in in-vivo cardiac functional findings are consistent with diastolic dysfunction resulting in the syndrome of HFpEF 28 , a condition commonly seen in COPD-Emphysema patients.

Myocardial hypertrophy without fibrosis

Post-mortem histological analysis revealed no sign of increased myocardial or perivascular fibrosis in the ELA-LPS group (Fig. 3A). Cardiac hypertrophy was detected in ELA-LPS treated animals as indexed by an increase in cardiac mass (Fig. 3B) and cardiac myocyte cell surface area (Fig. 3C). These findings indicate left ventricular hypertrophy in the absence of cardiac fibrosis.

Altered cardiac myocyte function

The HFpEF syndrome is due to impeded diastolic ventricular filling. Since the histological analysis excluded contribution of cardiac fibrosis in the stiffening of the heart (Fig. 3A), we next focused on the potential role of cardiac myocyte function in the observed syndrome. Membrane intact cardiac myocytes were isolated from Ctrl and ELA-LPS animals and ex-vivo functional tests were performed using an IonOptix cell system. The results are presented in Figure 4. Measurements on membrane intact and unloaded isolated cardiac myocytes revealed enhanced cellular Ca 2+ homeostasis in the ELA-LPS group. This was witnessed by increases in the basal level of [Ca 2+ ] (Fig. 4B), Ca 2+ transient (Fig. 4C), and Ca 2+ reuptake rate (Fig. 4D). The alteration in cellular Ca 2+ homeostasis was accompanied by cellular SL lusitropy (kinetics) (Fig. 4H), but not by enhanced SL shortening (Fig. 4G) or reduction in basal SL (Fig. 4F). These results may be indicative of increased adrenergic tone in exacerbated emphysema animals. However, the enhanced Ca 2+ homeostasis did not affect cellular contractility as indexed by the magnitude of SL shortening. Hence, these cellular excitation-contraction results do not fully explain the in-vivo HFpEF syndrome that is characterized, among other factors, by impaired cardiac relaxation.

Altered cardiac myofilament function

The membrane intact isolated cardiac myocyte measurements summarized above take place, by design, under unloaded conditions where sarcomeres operate at lengths well below those encountered in-vivo and, moreover, with minimal myofilament force development. Hence, such measurements report, predominantly, on cardiac cellular Ca 2+ homeostasis, but not on in-vivo cellular contractile function. To overcome this limitation, we additionally directly

-adrenergic pathways

Our cellular Ca 2+ homeostasis results (Fig. 4) are suggestive of increased -adrenergic tone in exacerbated emphysema. This notion was confirmed by a blunted in-vivo response to dobutamine infusion (Fig. 6A) in the ELA-LPS group indicative of reduced adrenergic reserve. In addition, the levels of cardiac Troponin-I phosphorylation at Serines 22/23 increased in ELA-LPS animals. These residues form the down-stream -adrenergic pathway targets for this contractile protein (Fig. 6B). Increased circulating catecholamine levels are a common finding in heart failure.

Moreover, pharmacological treatment with -adrenergic receptor antagonists has been shown to be of benefit, at least in systolic heart failure. To evaluate whether -blockers may be beneficial, ELA-LPS rats were treated with Bisoprolol, a specific  1 -receptor antagonist, for 5-weeks, 1 day following exacerbated emphysema induction (Fig. 1A). In-vivo evaluation by echocardiography revealed a reversal of cardiac diastolic dysfunction (Fig. 6C), a recovery of the blunted dobutamine stress-test (Fig. 6A) and a statistical trend towards a reduction of PKA-induced phosphorylation of Troponin-I (Fig. 6B). Moreover, in-vivo cardiac contractility was not affected as demonstrated by a maintained LVEF (Fig. 6D). Similar findings were observed at the cellular level, as illustrated in Figure 7. β-blocker treatment impacted cardiac calcium homeostasis by reducing the amplitude of calcium released (Fig. 7C), slowing of calcium reuptake examined myofilament function in isolated attached and force developing permeabilized (skinned) cardiac myocytes as presented in Figure 5. Skinned myocytes were exposed to varying levels of activating [Ca 2+ ] (Fig. 5A). Maximum [Ca 2+ ] activated force generating capacity was slightly increased in the ELA-LPS group (Fig. 5B). In addition, myofilament Ca 2+ sensitivity, as indexed by pCa 50 , was significantly blunted in exacerbated emphysema animals (Fig. 5C). Another important parameter of myofilament function is the rate of cross-bridge cycling, which we indexed here by K tr . The K tr parameter reflects the rate of force redevelopment following a rapidly imposed release-re-stretch maneuver as illustrated in Fig. 5D. ELA-LPS skinned myocytes displayed a significant slowing of myofilament crossbridge cycling (Fig. 5E). Taken together, these myofilament measurements indicate a blunting of myofilament Ca 2+ activating properties and slowing of cross-bridge cycling that is expected to contribute to a reduced cardiac relaxation rate and a resulting diastolic dysfunction in-vivo.

(Fig. 7D) and a higher basal calcium level (Fig. 7B). These are alterations that are consistent with β-blocker treatment effects. That is, many of the membrane-intact cellular contractility and Ca 2+ homeostasis parameters reversed, at least partially, to values observed in the control group. Moreover, the depressed myofilament cross-bridge cycling rate, as indexed by the K tr parameter (Fig. 7G), reverted to values seen in control skinned myocytes.

Overall, these in-vivo and ex-vivo data suggest that  1 -specific receptor antagonists may be beneficial to combat HFpEF in COPD-exacerbated emphysema.

DISCUSSION

Experimental exacerbated emphysema

The first aim of this study was to assess the relevance of an experimental exacerbated-emphysema rat model for exploring cardiac dysfunction, as seen in COPD patients. Our experiments showed that multiple orotracheal instillations of elastase followed by a single final simultaneous instillation of LPS, and subsequent 5-week incubation period, is effective in inducing pathologies that resemble those seen in COPD-exacerbated emphysema: alveolar parenchymal destruction, alveolar enlargement, airflow obstruction, and exercise intolerance. Of note, FEV 1 , a common clinical COPD index, is not accessible in animals. Importantly, our experimental model, by design, exhibits only the chronic impact of inflammation due to LPS exacerbation, as evidenced by the lack of circulating cytokines and pulmonary white blood cells. This is the first time this type of elastase induced emphysema and exacerbation rat model is explored in depth in terms of the delayed impacts on cardiac function; that is, previous studies focused either on events during the acute phases (within hours) following elastase or LPS treatment, [START_REF] Oliveira Mv De | Endotoxin-induced emphysema exacerbation: A novel model of chronic obstructive pulmonary disease exacerbations causing cardiopulmonary impairment and diaphragm dysfunction[END_REF][START_REF] Ceelen | Differential regulation of muscle protein turnover in response to emphysema and acute pulmonary inflammation[END_REF][START_REF] Fonseca Lmc Da | Emphysema induced by elastase enhances acute inflammatory pulmonary response to intraperitoneal LPS in rats[END_REF] or only on pulmonary pathophysiology [START_REF] Kobayashi | A single dose of lipopolysaccharide into mice with emphysema mimics human Chronic obstructive pulmonary disease exacerbation as assessed by micro-computed tomography[END_REF] .

Five weeks following the last elastase-LPS instillation, we found chronic tachycardia, exercise intolerance, increased -adrenergic tone, reduced cardiac output, and cardiac hypertrophy. Most importantly, we also found signs of LV diastolic dysfunction, but preserved cardiac contractility by echocardiography. These are the well-established clinical signs of diastolic heart failure as specified in pre-clinical guidelines [START_REF] Roh | Heart Failure With Preserved Ejection Fraction: Heterogeneous Syndrome, Diverse Preclinical Models[END_REF] . Thus, we succeeded in creating an exacerbated emphysema small rodent model associated with HFpEF.

We found no evidence of right ventricular dysfunction by echocardiography, in contrast to previous acute studies [START_REF] Oliveira Mv De | Endotoxin-induced emphysema exacerbation: A novel model of chronic obstructive pulmonary disease exacerbations causing cardiopulmonary impairment and diaphragm dysfunction[END_REF][START_REF] Oliveira | Characterization of a mouse model of emphysema induced by multiple instillations of low-dose elastase[END_REF] . This could be related to acute versus chronic impacts. Moreover, we deliberately used relatively low doses of elastase to allow for study of the chronic impacts of emphysema. Nevertheless, it is possible that subtle alterations in RV function were below the detection limit of right-sided echocardiography in rats and this would be the scope of further explorations of this experimental model. The cardiac phenotype was explored 5 weeks after the last instillation to better fit with the existing literature of the ELA-LPS model [START_REF] Henriques | Comparison between variable and conventional volumecontrolled ventilation on cardiorespiratory parameters in experimental emphysema[END_REF] . It will be interesting to explore cardiac function later after this period to verify if the heart decompensates on both sides.

A previous publication classified the level of increase in mean linear intercept to the severity of emphysema [START_REF] Thurlbeck | Internal surface area and other measurements in emphysema[END_REF] . According to their classification, an increase of 54% of the mean linear intercept of ELA-LPS animals suggests that our animals exhibited moderate to severe emphysema. Emphysema has been shown to be accompanied by lung hyperinflation that could induce "mechanical" diastolic dysfunction, mimicking a gaseous tamponade 6,[START_REF] Jörgensen | Effects of Lung Volume Reduction Surgery on Left Ventricular Diastolic Filling and Dimensions in Patients with Severe Emphysema[END_REF] , either caused by reduced LV filling and, thus, reduced myocardial preload [START_REF] Smith | Impaired left ventricular filling in COPD and emphysema: Is it the heart or the lungs?: The multi-ethnic study of atherosclerosis COPD study[END_REF] , or caused by elevation of pulmonary arterial pressure/resistance [START_REF] Sato | Pulmonary haemodynamics in awake rats following treatment with endotracheal pancreatic elastase[END_REF] or right ventricle pressure [START_REF] Lüthje | Exercise intolerance and systemic manifestations of pulmonary emphysema in a mouse model[END_REF] . De Oliviera et al [START_REF] Oliveira Mv De | Endotoxin-induced emphysema exacerbation: A novel model of chronic obstructive pulmonary disease exacerbations causing cardiopulmonary impairment and diaphragm dysfunction[END_REF] , employing a similar rat model of emphysema, showed pulmonary hyperinflation induced by emphysema as assessed by micro-computed tomography. Thus, our experimental animals most likely exhibited some pulmonary hyperinflation, and this still needs to be quantified. Therefore, pulmonary vessels [START_REF] Smith | Impaired left ventricular filling in COPD and emphysema: Is it the heart or the lungs?: The multi-ethnic study of atherosclerosis COPD study[END_REF] and atrial abnormalities could also play a role in the current exacerbated emphysema experimental animal model that needs to be further explored.

Inflammation and oxidative stress are major mechanisms known to contribute to COPD and HFpEF that may affect cardiac function. We did not find significant signs of circulating determinant of inflammation 5 weeks following induction of exacerbated emphysema, but for increased circulating IL-13 levels and a tendency for increased IL-6, IL-4 and IL-1B that did not reach statistical significance (p=0.064, p=0.076 and p=0.21, respectively). Oliveira et al [START_REF] Oliveira Mv De | Endotoxin-induced emphysema exacerbation: A novel model of chronic obstructive pulmonary disease exacerbations causing cardiopulmonary impairment and diaphragm dysfunction[END_REF] used a similar rat model but followed a different protocol: emphysema induced (4xELA at 2UI), 5 week incubation, followed by a final LPS instillation, and analysis after 24h. They observed in the lungs an elevation of the levels of cytokine-induced neutrophil chemoattractant-1, interleukin (IL)-1β, tumor necrosis factor-α, IL-6, and vascular endothelial growth factor in lung tissue, collagen fiber deposition in alveolar septa, airways, and pulmonary vessel walls, and increased dynamic lung elastance. In their study, inflammation in the lungs was associated with depressed LV systolic cardiac function as indexed by diminished LVEF. LV diastolic function, however, was not assessed. In contrast, we did not find significant modifications in pulmonary white blood cell content. However, it should be noted that in our experimental model, measurements were made well beyond the acute phase of inflammation, most likely after the "inflammation storm" induced by LPS instillation. Hence, the absence of elevated pulmonary white blood cells in lungs and plasma cytokines does not exclude inflammation induced cardiac dysfunction in this model. This could be explored by studying the extent of inflammation and cardiac dysfunction between the acute phase (LPS instillation) and the end of the protocol (i.e. 5 weeks incubation).

Altered cardiac myocyte function in exacerbated emphysema.

We did not find evidence for increased myocardial collagen deposits nor cardiac fibrosis. This absence of fibrosis, although uncommon, has already been found in other experimental HFpEF models [START_REF] Hegemann | Rightventricular dysfunction in HFpEF is linked to altered cardiomyocyte Ca2+ homeostasis and myofilament sensitivity[END_REF] and could be due to the relatively young age of the animals that we studied. We observed a modest but significant decrease in collagen content in ELA-LPS animals. The collagen content is calculated as the ratio of collagen area to collagen area plus myocyte area. Since the cell surface of the myocytes increased by 15% this could affect the relative content of collagen and may be responsible for the apparent relative decrease of collagen content. Therefore, it is unlikely that cardiac fibrosis underlies the diastolic dysfunction seen in our experimental exacerbated emphysema rat model. ELA-LPS treatment resulted in enhanced cellular Ca 2+ homeostasis closely resembling features of cardiac adrenergic stimulation such as increased basal cellular [Ca 2+ ] and magnitude of the Ca 2+ transient, concomitant with accelerated Ca 2+ kinetics. Indeed, evidence for increased circulating catecholamines included chronic tachycardia, a blunted dobutamine chronotropic response, and contractile protein Troponin-I PKA mediated phosphorylation. The cellular Ca 2+ homeostasis alterations were mirrored in SL mechanical parameters, with the notable exception of the extent of SL shortening. The latter is the cellular equivalent of whole heart ejection fraction and, as such, indicates that despite the increased Ca 2+ transient, cellular contractility was not affected. This implicates modification of the cardiac myofilament contractile protein's response to activating Ca 2+ . Indeed, direct examination of cardiac myofilament properties employing permeabilized cardiac myocytes confirmed this hypothesis. That is, both myofilament Ca 2+ sensitivity and cross-bridge cycling rate were found to be reduced in the ELA-LPS group. While, a decrease in myofilament Ca 2+ sensitivity following -adrenergic stimulation is well established [START_REF] Solaro Rj | Phosphorylation of troponin I and the inotropic effect of adrenaline in the perfused rabbit heart[END_REF] , a decrease in the cross-bridge cycling rate is not [START_REF] Biesiadecki | The troponin C G159D mutation blunts myofilament desensitization induced by troponin I Ser23/24 phosphorylation[END_REF] . This is likely the result of cardiac pathology, e.g. left ventricular hypertrophy secondary to chronic adrenergic stimulation. Our results suggest that novel treatment strategies targeting the cardiac myofilament may be of particular benefit in COPD-Emphysema.

-adrenergic pathways

The finding of evidence for increased levels of circulating catecholamines in exacerbated emphysema animals prompted us to explore possible benefits of chronic β-antagonist therapy. Chronic β-adrenergic stimulation has been shown to induce HFpEF in experimental animal models [START_REF] Dhot | Overexpression of endothelial β3-adrenergic receptor induces diastolic dysfunction in rats[END_REF] . There is an extensive literature focused on βblocker therapy in COPD and/or HFpEF patients with meta-analysis and registry [START_REF] Lund | Association between use of βblockers and outcomes in patients with heart failure and preserved ejection fraction[END_REF][START_REF] Hernandez | Clinical Effectiveness of Beta-Blockers in Heart Failure[END_REF] , or randomized controlled clinical trials [START_REF] Flather | FASTTRACK Randomized trial to determine the effect of nebivolol on mortality and cardiovascular hospital admission in elderly patients with heart failure (SENIORS)[END_REF][START_REF] Conraads | Effects of the long-term administration of nebivolol on the clinical symptoms, exercise capacity, and left ventricular function of patients with diastolic dysfunction: Results of the ELANDD study[END_REF] . However, clinical trials have mainly focused on safety outcomes concerning pulmonary function and exacerbation risk, or were designed to test acute exacerbation treatment scenarios [START_REF] Dransfield | Metoprolol for the Prevention of Acute Exacerbations of COPD[END_REF] . Indeed, to date persistent controversies remain regarding β-blocker therapy use in COPD patients [START_REF] Macnee | Beta-Blockers in COPD -A Controversy Resolved[END_REF] .

In the current study, we demonstrated reversal of both HFpEF signs and cellular alterations in response to chronic  1 -specifc receptor antagonist treatment in ELA-LPS animals. The cellular and molecular mechanisms underlying this beneficial impact are not entirely clear but may relate to blunting of chronic cellular Ca 2+ homeostasis "overload" leading to the syndrome of diastolic heart failure. Clarification of these mechanisms will require further investigations.

Limitations

It should be noted that our model explores the impact of chronic β 1 -blocker therapy in the absence of one of the main therapeutic approaches employed in COPD, β 2 agonists. These agents could exacerbate detrimental outcomes in an adrenergic overstimulation environment such as the one we described for our model. Like most, if not all, previous published studies on emphysema we have used only male rats to avoid physiological sex differences including hormonal variations. In many disease models, males are more sensitive to stress due to the absence of female hormones (i.e. cardiotoxicity of anthracyclines [START_REF] Moulin | Sexual Dimorphism of Doxorubicin-Mediated Cardiotoxicity[END_REF] ). This seems to be also the case for emphysema, since the experimental induction of emphysema in developing rats was prevented by daily injections of progesterone or medroxyprogesterone [START_REF] Ino | Cardiopulmonary effects of progestational agents in emphysematous rats[END_REF] . Sex impact in patients and experimental animal models have been investigated mostly with regard to cigarette smoking, where the sex appears to affect cigarette smoke detoxification by cytochrome P450. Accordingly, we cannot exclude the impact of sex on the outcomes of the current investigation.

The chronic cigarette smoke exposure model has been considered as a major component of COPD natural history and the "gold standard" for the animal experimental model of COPD. However, reported clinical studies highlight the existence of many non-smokers COPD patients, especially in the young [START_REF] Stolz | Towards the elimination of chronic obstructive pulmonary disease: a Lancet Commission[END_REF] . Moreover, cigarette smoke exposure in rodent models have shown limited pathophysiological impacts, equivalent to mild or moderate COPD GOLD stages I and II [START_REF] Wright | Animal models of chronic obstructive pulmonary disease[END_REF][START_REF] Churg | Everything Prevents Emphysema[END_REF] . In the context of our study, therefore it would be of great interest to evaluate LV dysfunction in a combined model with cigarette smoke and elastase exposure in the future.

The study of TnI phosphorylation in the group treated by β-blocker may have been insufficiently powered to achieve statistical significance. Anyway, the trend supports our hypothesis still.

Conclusions

Experimental exacerbation-emphysema was associated with exercise intolerance and diastolic heart failure due to increased -adrenergic tone and subsequent cardiac cellular and myofilament dysfunction. Chronic selective  1 receptor antagonist treatment reversed many of these impacts. Our results suggest that novel treatment strategies involving selective -receptor subtypes and targeted to the cardiac myofilament may be beneficial to combat exacerbated emphysema associated HFpEF. of the Montpellier University Hospital for access to the histologic platform and S. Lehmann and C. Hirtz for accessing to the PPC platform (IRMB, Montpellier, France). Under gaseous anesthesia Isoflurane (2.5%/O2), emphysema was induced in seven weeks old male rat (Janvier Labs, France) by oro-tracheal instillation of pancreatic porcine elastase (ELA, 4 units/200µL saline solution, Sigma-Aldrich, France) once a week for four weeks. To mimic COPD exacerbations, animals received with last ELA installation, lipopolysaccharide (LPS, E. coli O55:B5, 2.5mg/kg, Sigma-Aldrich, France). Animals treated the same way without receiving instillations served as controls (Ctrl) (Figure 1A). As a regulatory requirement, one buprenorphine injection (0.05 mg/kg) was given to avoid pain just after instillation. Animals were investigated 5 weeks after last instillation. One group of ELA-LPS animals received bisoprolol, a β1 receptor-specific blocker (2.5mg/kg/day in drinking water, bisoprolol hemifumarate, MedChemExpress, USA) from day 1 after the last ELA-LPS instillation until sacrifice.

Figure legends:

Histological characterization of emphysema, fibrosis and inflammation

At the end of the protocol, the lungs and heart were rapidly harvested after anesthesia (140 mg/kg sodium pentobarbital, i.p.) and total loss of consciousness. The lungs were inflated to formol for histology analysis. The heart was directly immersed in 4% formol. Lungs lobes were separated after formol fixation. Organs were properly rinsed, dehydrated in progressive ethanol baths, and embedded in paraffin. Six micrometers thick lung tissue sections apposed on slides were then stained with hematoxylin and eosin to assess the alveolar morphology organization. Emphysema was characterized using mean linear intercept (Lm) method with an Olympus microscope, 5X magnification. Briefly, using CellP program (Olympus), a grid was applied on the field with a 50µm spacing between lines. The software automatically detects the intercepts between the grid and alveoli, then the number of intercepts was divided by the total length 1 .

Automated red picrosirius staining was performed on 6µm cardiac tissue sections (Bio Optica red picrosirius picrate, Italy) at the histologic platform of the Department of Pathology of the Montpellier University Hospital. Stained slides were scanned with a slide scanner (Nanozoomer, Hamamatsu, Japan). Analysis of myocardial and perivascular tissue content in collagen was performed using ImageJ software (Image J, USA) and fibrosis area was measured automatically with the MRI Fibrosis Tool (https://github.com/MontpellierRessourcesImagerie/imagej_macros_and_scripts/wiki/MRI_ Fibrosis_Tool, last access on 20 April 2022). The person who performed the automatic analysis was blinded on the identity of the images. For each heart, we obtained 12 data points. We analyzed 2 different sections from the basal part of the heart and 2 different sections for the apical part. In each section of the heart we analyzed 3 regions in the freewall corresponding of the sub-epicardium/mid-wall and sub-endocardium. Eight perivascular sections per slide were randomly selected to measure percentage of red coloration over total area section. The areas that did not contain any tissue (inside the vessels or damages parts of the section) were excluded from the total area using the same modified tool.

After deparaffinization and rehydration, tissue slides were subjected to antigen retrieval by pepsin. Lung sections were stained for neutrophils and eosinophils with vectastain kit (vector laboratories). Briefly, after peroxidase and alkaline phosphatase activity blocking with bloxall (vector laboratories), sections were blocked with 10% normal horse serum 10 min following by 1 hour incubation with a mouse anti human eosinophil major basic protein antibody (Biorad), or a rabbit anti human neutrophil elastase antibody (abcam). After rinsing a biotinylated secondary anti mouse rat adsorbed IgG antibody (vector laboratories) or biotinylated anti rabbit IgG antibody (vector laboratories) was added for 30 minutes, followed by the addition of a peroxidase/streptavidin complex (VECTASTAIN universal kit; Vector Laboratories, Peterborough, United Kingdom). Diaminobenzidine (vector laboratories) was used to visualize the antigen of interest. Sections were counterstained using hematoxylin.

Stained slides were scanned with a slide scanner (Nanozoomer, Hamamatsu, Japan).

Quantification of the number of neutrophils and eosinophils was performed by cumulative counts on 5 fields of bronchiole or alveola or vessel view per section. For bronchiole, neutrophil and eosinophil number was expressed in number/µm of perimeter of bronchiole.

For alveola, one field is a square of 0.407mm² and neutrophil and eosinophil were expressed as number/mm². For vessels, they were expressed in number/µm of perimeter of internal adventitia.

Maximal oxygen consumption (V'O 2 max)

The maximal oxygen consumption (V'O 2 max) is measured by an incremental exercise test (EFX) performing on a metabolic treadmill coupled to a gas analyzer system (Oxymax, Columbus Instruments, Columbus, USA). The implemented protocol was adapted from Musch et al 2 . A first habituation phase, lasting 5 days, was carried out in the animals, one week before the test carried out 3 .

During the EFX the rat have a period of acclimation of 5 min at rest, then the treadmill protocol starts with a speed of 10 m/min and gradual increase in speed in steps of 5m/min every 3 minutes until the animal is exhausted. Exhaustion was defined as the animal remaining on the electrical stimulation grid without attempting to re-engage the treadmill within 15 seconds.

The V'O 2 and V'CO 2 flow rates are calculated based on the measurement of the fractions in O 2 (FO 2 , in %) and in CO 2 (FCO 2 , in %) at the inlet and at the outlet of a sealed chamber every 30 seconds (Oxymax, Columbus Instruments). V'O 2 max was defined as the higher value of V'O 2 obtained (average of 30 seconds) before stopping the effort.

Western blot analysis

PKA dependent myocardial protein phosphorylation was studied, as previously described 4 .

Briefly, frozen LV tissue powdered using tissue grinder on dry-ice and solubilized in nonreducing Laemmli buffer. Proteins were separated using gradient SDS-PAGE electrophoresis and transferred onto membranes (nitrocellulose, GE Healthcare, USA). Membranes were blocked with the Odyssey Blocking Buffer in TBS (LI-COR Biosciences, USA) and incubated overnight with primary antibodies. Bands were revealed and quantified with the Odyssey system (LI-COR Biosciences, USA) after incubation with fluorescent secondary antibodies.

Phosphorylation level was expressed relative to total protein content, and protein content was normalized to GAPDH content.

Plasma multiplex cytokine analysis

Six plasma cytokines (IL-1β, IL-4, IL-6, IL-10, IL-13 and KC/GRO) were measured using chemiluminescence-based assays from Meso Scale Discovery (MSD, Gaithersburg, MD, USA).

For the 6 cytokines, a V-PLEX Pro-Inflammatory Panel 2 Rat Kit was used (MSD, Gaithersburg, MD, USA). The detection ranges are IL-1β 6.92-8100 pg/mL, IL-4 0.69-723 pg/mL, IL-6 13.8-8550 pg/mL, IL-10 16.4-15 700 pg/mL, IL-13 1.97-1080 pg/mL, and KC/GRO 1.04-728 pg/mL.

All assays were performed in duplicate. Analyses were done using a QuickPlex SQ 120 instrument (MSD, Gaithersburg, MD, USA) and DISCOVERY WORKBENCH® 4.0 software. All samples were run at the same time.

In vivo respiratory function

Respiratory function was analyzed the day before sacrifice using a whole-body plethysmograph (Emka Technologies, France) and the Iox2 software (Emka Technologies, France) as previously described 5 . Animals were first familiarized with the chamber the week before the test.

In vivo cardiac function by echocardiography and ECG telemetry

In vivo contractile cardiac function was explored using an echocardiograph (Vevo 3100, Visualsonics, Canada) with a 20 MHz probe as previously 6 . Animals were anesthetized with Isoflurane (2.5 -3.5%) under 1L/min air. Particular attention has been paid to ensure that the heart rate was the same in all groups to allow proper comparison of the echocardiography data, in bpm: ctrl (358 ± 4.9), ELA-LPS (355 ± 6.0), and ELA-LPS+β-blocker (357 ± 14.9).

Following ESC working group position paper For pharmacological testing with dobutamine (2mg/kg, i.p), ECG was recorded 4 hours before and 4 hours after the injection. Each dobutamine challenge was continuously followed through automatic RR detection, from which the data describing the most profound changes were chosen for the analysis of cardiac reserve. These changes were selected from the most significant decrease in RR intervals and results (mean over at least 8 000 cardiac cycle PQRST)

were compared between pre-dosing and post-dosing periods.

In vivo non-invasive systemic pressure measurement

Non-invasive systemic pressure was measured by a tail-cuff apparatus (Coda® High throughput system, non-invasive pressure system, Kent Scientific, USA) under temperaturecontrolled conditions (25°C). After a week of familiarization to the procedure, animals were placed inside the system, and recorded during 15 minutes with maximal occlusion pressure of 250 mmHg. Five good quality recodings were used to mean the pressure.

Cardiac excitation-contraction coupling

Intact cardiomyocytes were isolated from the heart by enzymatic digestion as previously described 9 . Briefly, the heart was harvested while beating and quickly cannulated on a Langendorff apparatus by the aorta. The heart was first washed with a calcium (Ca

Permeabilized cardiomyocytes and mechanics

Permeabilized cardiomyocytes were isolated by mechanical dissociation as described previously 10 . The heart was flushed of its blood and the left ventricle was dissected, quickly frozen in liquid nitrogen, and stored at -80°C until use. The day of experiment, about 20 mg of tissue was pre-permeabilized in a relaxing solution at [Ca 2+ ]=10 -9 M with anti-protease cocktail (PMSF, Leupeptine, E64) and 1% of Triton X100 within 5 minutes. Tissue was mechanically crushed using a polytron (Ultraturax®) for 2-3 seconds. After filtration, the myocytes were further permeabilized in 0.3% Triton X-100 solution for 6 min at room temperature to remove remaining sarcolemma membranes. After extensive washing with ice-cold relaxing solution, cells were kept on ice and used within the day. Cells were attached to metal tips with an optical glue (NOA 63, North Brunswick, USA) polymerizing under UV light. One tip was linked to a force transducer (AE801, SensoNor, Horten, Norway) allowing to measure developed tension in isometric conditions. The other tip was connected to a piezoelectric motor (ThorLabs Inc, USA) used to shortly modify the length cell for the measure of actin-myosin bridges reformation (kTr). The relationship between Ca 2+ -activated force and internal Ca 2+

concentration was measured at two different sarcomere length (SL): 1.9 µm and 2.3 µm.

Curves were fitted using a modified Hill equation with GraphPad Prism v8.

Statistical analysis

Data are presented as mean± SEM. Differences were assessed using Student's t-test or Mann Whitney's rank test whenever distribution was not Gaussian after Shapiro-Wilk's normality test. A p-value inferior to 0.05 was considered as significant using GraphPad Prism V9.0 software (GraphPad Software, USA). Graphs were realized on GraphPad Prism v9.0.

Figure 1 :

 1 Figure 1: Chronic emphysema rat model with exacerbation.

Figure 2 :

 2 Figure 2: Echocardiography indicates HFpEF in ELA-LPS rats.

Figure 3 :

 3 Figure 3: Myocardial hypertrophy without fibrosis in ELA-LPS rats.

( B )

 B Heart weight to body weight ratio; n=44 Ctrl and 18 ELA-LPS animals. (C) Membrane intact isolated left ventricular myocytes cell surface area; n=20 cells per animal, 5 animals per group.

(

  A) and (C) are represented as median with total distribution and 25 -50% interquartile. (B) is represented as mean ± SEM. Differences were assessed by Mann-Whitney test *p<0.05.

Figure 4 :

 4 Figure 4: Ex-vivo altered myocardial cellular function.

Figure 5 :

 5 Figure 5: Ex-vivo altered cardiac myofilament function.

Figure 3 A

 3 Figure 3

  After 30 min stabilization, each animal was visually monitored and ventilatory parameters were recorded during inactive phase for 15 min under two experimental conditions. Animals were exposed to room air (1.5L/min inflow). The parameters measured were inspiration time (Ti), expiration time (Te), minute-volume (MV), tidal-volume (TV), peak inspiratory flow (PIF), peak expiratory flow (PEF), expiratory flow 50 (EF50), relaxation time (RT), frequency of breathing (f), enhanced pause (PenH) and EV (expired volume).

  2+ )-free extracellular physiological solution and then with an enzyme-containing solution (Collagenase type IV, Worthington) during 25 to 35 minutes. The free wall, septum and right ventricle were separated, and digestion was stopped with bovine sodium albumin. The tissue was mechanically disrupted with a pipette to release the myocytes. The extracellular Ca2+ concentration was progressively raised to 1mM.Cardiomyocytes were loaded with the ratiometric Ca 2+ dye indo-1AM at room temperature during 20 min (2 µM, Life technologies, St-Aubin, France) and cell shortening/Ca 2+ transients were recorded using electrical-field stimulation (1 Hz, 2 msec, 10V). Experiments were done in Tyrode solution (140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 20 mM HEPES, 1.8 mM CaCl 2 and 11 mM glucose, pH 7.4). Sarcomere length (SL) and fluorescence wavelengths emitted at 405 nm (F 405 ) and 480 nm (F 480 ) were simultaneously recorded using the IonOptix®system (Milton, USA) coupled to a Zeiss micro-scope (Zeiss, 40× oil). Data were analyzed using IonWizard 6.6 software.

Supplemental Figure 1 :

 1 as mean±SEM. (n=number of cells/number of animals); pCa 50 Ca 2+sensitivity of isometric-force production, Tmax maximum Ca 2+ -activated active tension Tpassive Ca 2+ -independent passive tension, ktr,sub-maximal and maximal rate constant of force redevelopment, nH Hill steepness of the force-pCa curve characterizing the cooperativity between myofilament units, p<0.05 *ELA-LPS vs Ctrl † ELA-LPS vs ELA-LPS+ βblocker. The p-value column corresponds of the comparison ELA-LPS vs Ctrl. (A-H) Pulmonary inflammation. Eosinophil (A) and neutrophil (E) immunohistochemistry staining on pulmonary sections, X40. White blood count per µm of bronchiole for neutrophils (B) and eosinophils (F); per mm 2 of lung parenchyma for neutrophils (C) and eosinophils (G); per mm of vascular internal diameter for neutrophils (D) and eosinophils (H). (n=4-5 animals/group, 6 areas per animals). (I-N) Systemic plasmatic inflammation cytokines with predominantly T-helper 1 response with (I) Interleukin 6 (IL-6) (J) keratinocytes-derived chemokines (KC/GRO) and (K) Interleukin-1beta (IL-1β) and predominantly T-helper 2 response with (L) Interleukin 4 (IL-4), (M) Interleukin-10 (IL-10) and (N) Interleukin-13 (IL-13). (n=6 animals per group, duplicate). Data are represented as mean ± SEM) Graphical abstract

  7 , long axis B and M-mode were acquired in

	parasternal long axis. Short axis B, M-mode, 4 cavity chamber view, Doppler and tissular
	Doppler were acquired in parasternal short axis. Aortic and pulmonary artery view were
	acquired in modified parasternal short axis.
	For electrocardiogram (ECG) recordings, rats were equipped with telemetric transmitter (CA-
	F40, Data Sciences International, St. Paul, MN, U.S.A) under general anesthesia (Isoflurane,
	2.5% in O 2 ) and allowed to recover for 15 days 8 . Rats were monitored with ECG recordings by
	using a signal RCP-1 receiver connected to a data acquisition system (Ponemah Physiology
	Platform v6.42, DSI). The basal ECG i.e. without forced-exercise were collected continuously
	over 24 h at a sampling rate of 2 kHz in a calm and quiet room. Successive RR interval series
	were obtained using Ponemah Physiology Platform from 8 hours ECG recording, taking in the
	same time slot for all animals (10:00 PM to 06:00 AM). PR, QRS and QT intervals were
	determined from the same ECG segment.

Table 2 :

 2 In-vivo electrical function measured by electrocardiogram telemetry five weeks after the last ELA instillation during a normal activity in their cage.

	Supplemental Table 1: Plethysmography and lung morphology
		Ctrl	ELA-LPS	p-value
	n	13	15
	Ti (msec)	229 ± 10.8	225 ± 8.5	0.767
	Te (msec)	403 ± 19	446 ± 22	0.279
	TV norm (mL/kg)	4.1 ± 0.13	4.4 ± 0.22	0.311
	MV norm (mL/min/g)	0.39 ± 0.02	0.40 ± 0.01	0.910
	PIF (mL/s)	14.4 ± 0.7	14.6 ± 0.9	0.808
	PEF (mL/s)	9.6 ± 0.4	8.4 ± 0.2*	0.019
	Lm (µm)	48 ± 1	68 ± 3*	<0.001

Results are presented as mean±SEM. Ti: inspiration time; Te: expiration time; TV: tidal volume; MV: minute volume; PIF: peak inspiratory flow; PEF: peak expiratory flow ; Lm: mean linear intercept. *p<0.05. The p-value column corresponds of the comparison ELA-LPS vs Ctrl. Results are presented as mean±SEM. HR: heart rate (beat per minute); Dobu HR: heart rate variation after dobutamine injection; p<0.05 *ELA-LPS vs Ctrl ; †ELA-LPS vs ELA-LPS+ βblocker. The p-value column corresponds of the comparison ELA-LPS vs Ctrl. Supplemental

Table 3 :

 3 In-vivo cardiac contractile function five weeks after the last ELA Results are presented as mean±SEM. bpm: beat per minute HR: heart rate; LVEF: left ventricular ejection fraction; FS: fractional shortening; CO: cardiac output; E: passive mitral inflow wave; A: active mitral inflow wave; e': early diastolic mitral annular velocity; LVID: left ventricular internal diameter in diastole (d) and systole (s); AWT: anterior wall thickening;

	instillation
		Ctrl	ELA-LPS	p-value	ELA-LPS + β-
					blocker
	n	22	19		9
	LV function
	Heart rate (bpm)	358.5 ± 5.0	355.6 ± 6.0	0.712	357.2 ± 14.9
	LVEF (%)	61.7 ± 1.5	64.0 ± 1.9	0.334	66.1 ± 2.4
	FS (%)	18.9 ± 1.4	16 .8 ± 1.1	0.267	19.1 ± 2.0
	CO (mL/min)	132 ± 5	116 ± 6*	0.048	112 ± 5
	E (mm/s)	663 ± 21.7	708 ± 38.7	0.300	684 ± 44.5
	A (mm/s)	738 ± 22.3	675 ± 40.4	0.169	682 ± 48.2
	e' (mm/s)	18.8 ± 1.7	13.2 ± 1.0*	0.008	26.7 ± 4.4 †
	E/A	0.91 ± 0.04	1.08 ± 0.05*	0.015	1.02 ± 0.06
	E/e'	39.3 ± 2.8	58.2 ± 5.0*	0.002	34.5 ± 7.8 †
	LV morphology
	LVIDd	7.7 ± 0.2	7.4 ± 0.1	0.140	6.9 ± 0.3
	LVIDs	3.7 ± 0.2	3.4 ± 0.1	0.548	3.1 ± 0.3
	AWT	73.2 ± 5.1	75.0 ± 5.2	0.810	81.2 ± 8.2
	PWT	56.9 ± 4.3	70.4 ± 7.2	0.098	51.8 ± 14.0
	IVS wall thickening	68.6 ± 5.3	72.2 ± 5.9	0.656	66.6 ± 6.3

PWT: posterior wall thickening; IVS: interventricular septum; TAPSE: tricuspid annular plane systolic excursion; S tricuspid: systolic tricuspid annular velocity; PET: pulmonary ejection time. p<0.05 *ELA-LPS vs Ctrl ; † ELA-LPS vs ELA-LPS+ β-blocker. The p-value column corresponds of the comparison ELA-LPS vs Ctrl. Supplemental

Table 4 :

 4 Excitation-contraction coupling of intact cardiomyocytes from LV Results are presented as mean±SEM. (n=number of cells/number of animals); p<0.05 *ELA-LPS vs Ctrl † ELA-LPS vs ELA-LPS+ β-blocker. The p-value column corresponds of the comparison ELA-LPS vs Ctrl.

		Ctrl	ELA-LPS	p-value	ELA-LPS + β-
					blocker
		(n=441/11)	(n=370/9)		(n=197/5)
	SL shortening
	diastolic (µm)	1.75 ± 0.003	1.76 ±0.003*	0.0075	1.72 ± 0.004 †
	amplitude (%)	11.7 ± 0.16	12.01 ± 0.16	0.131	12.3 ± 0.23
	Contraction velocity (µm/s)	3.85 ± 0.06	4.07 ± 0.07*	0.006	3.30 ± 0.08 †
	Relaxation velocity (µm/s)	2.56 ± 0.05	2.77 ± 0.05*	<0.001	1.89 ± 0.05 †
	Ca 2+ transient
	diastolic	0.763 ± 0.004	0.783 ± 0.004*	<0.001	0.798 ± 0.005 †
	amplitude	0.272 ± 0.005	0.307 ± 0.005*	<0.001	0.277 ± 0.006 †
	Tau (ms)	245 ± 3	234 ± 3*	0.010	343 ± 7 †

Acknowledgments:

The authors thank Laurie Alburquerque from the Lab animal facility, the Imagerie du Petit Animal de Montpellier (IPAM) for access to high-resolution ultrasound and Montpellier Ressources Imagerie (MRI, BioCampus, CNRS) for access to the slide scanner. We also want to thank I. Serre and the Department of Pathology

Funding: This work was supported by the Fédération Française de Cardiologie to AB (#R20012FF).

Competing interests:

The authors have no disclosures to declare related to this paper, except for AB who has, within the two last years, received personal fees from GlaxoSmithKline, Regeneron, Chiesi, Teva, Boehringer Ingelheim; personal fees and other from AstraZeneca and Novartis.

Data availability statement:

The data underlying this article will be shared on reasonable request to the corresponding author.