MRI Assessment of Cardiomyopathy Induced by β1-Adrenoreceptor Autoantibodies and Protection Through β3-Adrenoreceptor Overexpression

The cardiopathogenic role of autoantibodies (aabs) directed against β1-adrenoreceptors (β1-AR) is well established. In mouse models, they cause progressive dilated cardiomyopathy (DCM) whose characterization with echocardiography requires prolonged protocols with numerous animals, complicating the evaluation of new treatments. Here, we report on the characterization of β1-aabs-induced DCM in mice using 11.7T MRI. C57BL/6J mice (n = 10 per group) were immunized against the β1-AR and left ventricular (LV) systolic function was assessed at 10, 18 and 27 weeks. Increase in LV mass/tibial length ratio was detected as the first modification at 10 weeks together with dilation of cavities, thereby outperforming echocardiography. Significant impairment in diastolic index was also observed in immunized animals before the onset of systolic dysfunction. Morphometric and histological measurements confirmed these observations. The same protocol performed on β3-AR-overexpressing mice and wild-type littermates (n = 8–12 per group) showed that transgenic animals were protected with reduced LV/TL ratio compared to wild-type animals and maintenance of the diastolic index. This study demonstrates that MRI allows a precocious detection of the subtle myocardial dysfunction induced by β1-aabs and that β3-AR stimulation blunts the development of β1-aabs-induced DCM, thereby paving the way for the use of β3AR-stimulating drugs to treat this autoimmune cardiomyopathy.

MRI cardiac function evaluation upon induction of β1AR aabs. After 10 weeks of follow-up, mice immunized against the β 1 AR-EC II already showed a statistically significant increase in the LV/TL ratio compared to control mice (4.2 ± 0.1 vs 3.8 ± 0.1 mg/mm, p < 0.05) (Fig. 2, left panel and Table 1). Over time, a continuous increase of this parameter was observed in the β 1 -immunized group, accompanied by signs of progressive dilation of LV (Fig. 2, left panel and Table 1). These MRI data for LV mass and LV/TL were confirmed by ex vivo measurements at 28 weeks (Table 1).
Analysis of the diastolic index showed an impaired diastolic function in the treated group from 18 weeks that become significantly altered at 27 weeks (51.4 ± 1.1 vs 63.1 ± 4.3%, P < 0.05) (Fig. 2, right panel and Table 1). By contrast, systolic function assessed both by EF and systolic index remained within normal values in the immunized group (Table 1).
We also performed qPCR analyses to identify changes in mRNA expression from corresponding cardiac tissues (Table 2). mRNA expression of β AR-1 and -2 (ADRB 1-2) remained unchanged between control and β 1 -immunized group while a two-fold increase in β AR-3 gene expression (ADRB3) was observed in the immunized group (although not reaching statistical significance, Table 2). No re-expression of the fetal gene program was found based on the levels of myosin heavy chains (MYH6 and MYH7 coding for the alpha and beta isoform, respectively) and natriuretic peptides (ANP and BNP for atrial and brain forms, respectively); a trend toward an increase in the BNP gene expression was however observed in β 1 -immunized mice (p = 0.07, Table 2). β3TG mice are protected from β1AR aabs-induced cardiomyopathy. We tested whether the overexpression of the β 3-AR could protect from developing β 1-aabs-induced DCM by applying the protocol of immunization previously described to 12 transgenic (heterozygous) mice overexpressing the β 3-AR (TG) and 12 wild-type littermates (WT). 5 mice died in each group upon active immunization against β 1 AR-EC II before the end of the protocol.
Cardiac MRI measurements after 27 weeks showed statistically significant differences between the two immunized groups: LV/TL ratio remained unaltered in immunized β 3 -overexpressing mice, with a value of Figure 2. MRI follow-up in β1-immunized (n = 7-9) and control mice (n = 10). The panels depict the time course of the following parameters: LV mass/tibial length ratio (LV/TL), end-diastolic volume (EDV) and diastolic index. Error bars indicate SEM. *P ≤ 0.0.05; **P ≤ 0.0.01; ***P ≤ 0.0.001; ****P ≤ 0.0.0001 vs control.    3.5 ± 0.2 mm/mg (vs. 4.3 ± 0.2 mm/mg in immunized wild-type animals, p < 0.05) (Fig. 5A, left panel). These data were confirmed by ex vivo LV/TL measurements after sacrifice of the animals (Fig. 5A, right panel). Interestingly, while determination of systolic indexes did not reveal any difference between mouse groups (Fig. 5B), evaluation of diastolic indexes reinforced the concept of β 3-AR-mediated protection. First, significant differences between the two immunized groups were observed as soon as 18 weeks (Fig. 5C). Second, while LV/TL ratio measurements only revealed a trend towards an increase in control littermates of transgenic animals (see two first sets of points in Fig. 5A graphs), diastolic indexes revealed a statistically significant reduction after 27 weeks, confirming the earliness of diastolic dysfunction in this DCM model (Fig. 5C). Other MRI measurements were not altered when compared to control groups after 27 weeks of follow-up (not shown).

Discussion
The major findings of this study are related to the application of UHF MRI technology to study the early development of β 1aabs-driven autoimmune cardiac dysfunction and to the identification of β 3-AR as key actor of a counterbalancing pathway preventing the occurrence of DCM in this model. Previous studies using echocardiography have reported the first anatomical changes at rest in β 1 -immunized BalbC 25 and C57BL/6J mice 26 at 25 weeks. In our study with C57BL/6J mice immunized against the β 1-AR, using UHF MRI, we were able to show early aabs-induced cardiomyopathic changes after only 10 weeks. LV hypertrophy (increase in LV/TL ratio) was detected before transition to the complete phenotype of DCM. Furthermore, we applied to our UHF MRI study a recently described method based on the concepts of echocardiographic color kinesis 41,42 in order to derive systolic and diastolic indexes in our animals. This led us to document that diastolic dysfunction was detectable before the occurrence of systolic dysfunction. These results open new perspectives for clinical investigations in patients carrying β 1-aabs. Indeed, several methods (i.e. echographic speckle tracking and MR tagging ref. 43) are now available besides the classical echocardiographic mitral inflow color Doppler (E/A) measurements in order to thoroughly investigate diastolic function in patients. If these data are confirmed by further clinical studies, appearance of diastolic dysfunction in β 1-aabs-positive patients could be used as an early marker of evolution to heart failure, requiring an intensification of treatments.
Besides the above technological benefits of UHF MRI applied to autoimmune DCM, we showed that transgenic mice harboring a cardiac-specific human β 3-AR transgene were protected from myocardial dysfunction induced by β 1-aabs. Although, unlike in C57BL/6J mice, only a trend to a cardiac dilation (ie, increased LV/TL ratio) was detectable in the immunized WT group (vs. non-immunized mice) at the end of our study, a significant reduction in the diastolic index confirmed the development of β 1aabs-mediated cardiac alterations in these mice. Different genetic backgrounds are likely to account for the LV/TL ratio discrepancy but this observation also emphasize the earliness of the diastolic dysfunction in this model of autoimmune DCM. Importantly, in β 3-AR overexpressing mice, the diastolic index was not decreased in response to immunization and was actually even slightly increased. Altogether, these data indicate a significant protective effect of β 3-AR overexpression against the development of heart damage induced by β 1-aabs (vs. immunized WT mice) and thereby open new therapeutic perspectives in the treatment of autoimmune DCM.
As β 3-AR have functionally opposite effects to β 1-AR and β 2-AR on cardiac muscle and are more resistant than β 1-AR and β 2-AR to homologous desensitization 33 , they represent attractive candidates for efficient pharmacological modulation in the diseased heart 44 . Recent data published by our team have already shown that β 3TG mice were protected against early cardiac dysfunction induced by neurohormone infusion (i.e. angiotensin II or isoproterenol) 37 . The present study confirms the potential beneficial effects of β 3-adrenergic stimulation to also prevent the development of autoimmune DCM. In addition to the attenuation of β 1-adrenergic inotropic responses, the nitric oxide production consecutive to a β 3-AR stimulation of microvascular endothelial cells 45 and cardiac myocytes 37 may help to maintain a normal left ventricular diastolic function during the early stages   of cardiac dysfunction; improving diastolic relaxation can indeed exert a beneficial hemodynamic effect through the maintenance of the Frank-Starling response 46 . The latter property seems even more relevant for the treatment of β 1-aabs-induced cardiomyopathy, in the light of the early diastolic dysfunction reported in mice immunized against β 1-EC II . A limitation in our study is the use of transgenic β 3 AR-overexpressing mice which per se means that this pathway is continuously influencing the cardiac phenotype of these animals and may thus prevent dysfunction from the earliest stages of its development. Still, these results pave the way for the evaluation of bona fide β 3-AR agonists or the preferred use of β -blockers endowed with such β 3-AR stimulatory activity in the treatment of autoimmune β 1-aabs-induced DCM. Nebivolol represents a third generation selective β 1-AR antagonist with ancillary metabolic effects involving a β 3-AR stimulation. Nebivolol could thus bring an increment of efficacy vs. conventional beta-blockers whose usefulness has already been demonstrated in the context of β 1-aabs-induced DCM (with however a lower efficiency than therapies using neutralizing peptides) 30 . We previously showed that nebivolol treatment of coronary microvessels led to NO-dependent vasorelaxation 47 while others documented a further protection of nebivolol administration against endothelial dysfunction though a net reduction in oxidative stress 48 . Altogether these studies suggest that the dual β 1/β 3-AR modulation, already demonstrated in previous studies for heart failure and myocardial infarction with Nebivolol 49 , could be extended to patients with β 1 -aabs-induced DCM.
In conclusion, we showed that UHF MRI allows the precocious detection of mouse myocardial remodeling induced by β 1-aabs, at earlier time-points than anticipated based on previous reports using echocardiography. Importantly, this technology allowed us to identify a diastolic dysfunction occurring before the onset of systolic dysfunction in this autoimmune cardiac disease and to provide evidence supporting the therapeutic potential of drugs endowed with beta-3 AR agonistic activity for the treatment of β 1-aabs-induced DCM. β3TG mice. Male mice harboring an α -myosin heavy chain promoter-driven human β 3-AR transgene generated as described previously 50 , were used to produce heterozygous β 3TG mice and wild-type littermate controls (n = 40); the original β 3TG mouse line had been selected to exhibit a moderate overexpression of the transgene (matching the ex vivo myocardial response to a β 3-AR agonist). Mice from β 3TG and wild-type groups were randomly distributed in two subgroups and immunized according to the protocol described above, except that subcutaneous injections were performed under light anaesthesia with isoflurane 2-3% in oxygen for 3 minutes, and that no retro-orbital blood sample was taken at the same time to minimize animal stress. 12 mice per group were immunized with the peptide while 8 mice per group receive the vehicle and were used as controls.

Methods
Morphometric measurements. At the end of the study, after a follow-up of 28 weeks, animals were euthanized by cervical disclocation and their left ventricular weight and tibial length were measured. Hearts and sera were collected for analysis and stored at − 80 °C.

MRI measurements. Cardiac MRI (CMR) acquisition.
Each animal was scanned at 10, 18 and 27 weeks of treatment on a 11.7T MRI scanner dedicated to small animal applications (Biospec, Bruker, Ettlingen, Germany). A quadrature 1 H resonator was used for radiofrequency transmission (inner diameter = 72 mm, length = 6.6 cm) in conjunction with a surface receive-only coil array (length = 10.7 cm). Anaesthesia was induced with 3% isoflurane in oxygen, and then maintained with 0.5-2% isoflurane during the entire procedure, in order to remain within physiological heart rates (around 500 heartbeats/min). Animals were placed in prone position and monitored for electrocardiogram and respiration with neonatal electrodes wrapped around the paws and a pneumatic sensor placed under the animal. The body temperature was followed by using a rectal probe and regulated with a dedicated heating blanket. Cardiac scout images were obtained in the conventional planes with a tripilot sequence. Then an Intragate 2D cine Fast Low Angle Shot (FLASH) sequence was applied to acquire a stack of seven to eight 1-mm thick contiguous short-axis images covering the entire ventricles, perpendicular to the LV long-axis. Imaging parameters: repetition time CMR Image analysis. The LV systolic function was assessed from the stack of short axis images by tracing epicardial and endocardial borders on Segment software (Medviso v1.8, Lund, Sweden). End-diastolic (EDV), end-systolic (ESV) and stroke volume (SV) were determined (μ l). LV ejection fraction (EF, as %) and LV mass (mg) were subsequently deduced. Systolic and diastolic indexes were determined as previously described 41,42 . Briefly, we visually determined the end-diastolic phase, end-systolic phase and the phase at 30% of diastole, and traced the endocardial contours at the mid-ventricular level on the Osirix imaging software (v4.0; Pixmeo; Geneva, Switzerland). Then we calculated the systolic fractional area change (systolic index, as %) through the following formula: EDA-ESA/EDA, where EDA = end-diastolic area and ESA = end-sytolic area. The fractional Scientific RepoRts | 7:43951 | DOI: 10.1038/srep43951 area change during the first 30% of diastole (diastolic index, as %) was also calculated through the following formula: dA-ESA/EDA-ESA, where dA = area at 30% of diastole. All analyses were performed on a blinded basis.
Cardiac histochemistry. At the end of the study hearts were excised and representative pieces of left ventricles were fixed in 4% formaldehyde after rinsing with saline. For analysis of cardiac fibrosis, 5-μ m sections of paraffin embedded hearts were stained with picrosirius red. Stained sections were digitalized with a SCN400 slide scanner (Leica Biosystems, Wetzlar, Germany). Quantification was made with Tissue IA software (Leica Biosystems, Dublin, Ireland). Area occupied by interstitial fibrosis was expressed as a percentage of total myocardial area. To quantify transverse cardiomyocyte area and capillary density, cryosections were stained with Wheat germ agglutinin (WGA) as a membrane marker, and with biotinylated-Isolectine B4 as an endothelial marker. Mounted slides were observed with an Axioimager Z1/Apotome microscope equipped with a MRM camera (Zeiss, Germany). The data analysis was performed with Axiovision software (Zeiss, Germany). A minimum of 40 to 100 cells from 9 sections were measured in each heart. Other heart cryosections were incubated with antibodies raised against the monocyte marker CD11b and the lymphocytic marker CD45 (rat anti-CD11b and rat anti-CD45, both from BD Bioscience, San Jose, CA). Slides were scanned with a MIRAX Scanner (Zeiss, Germany) and analyzed with FRIDA software (Johns Hopkins University, Baltimore, MD). All analyses were performed on a blinded basis.
Statistical Analysis. Data are expressed as mean ± SEM. Raw data were analyzed for normal distribution using the Shapiro-Wilk test. When normally distributed, unpaired Student t test was used to compare differences between two groups and one-way analysis of variance followed by Bonferroni post-hoc test was used to compare 3 or more groups. In the absence of normal distribution, the statistical significance of differences was tested with non-parametric tests (Mann-Whitney or Kruskall-Wallis followed by Dunn's test). P < 0.05 was considered statistically significant. Statistical analyses were performed with GraphPad Prism 5.04 (San Diego, CA) and JMP 11.2.0 (SAS Institute, Cary, NC).