Segmental analysis by speckle-tracking echocardiography of the left ventricle response to isoproterenol in male and female mice

We studied by conventional and speckle-tracking echocardiography, the response of the left ventricle (LV) to a three-week continuous infusion of isoproterenol (Iso), a non-specific beta-adrenergic receptor agonist in male and female C57Bl6/J mice. Before and after Iso (30 mg/kg/day), we characterized LV morphology and function as well as global and segmental strain. We observed that Iso reduced LV ejection in both male (−8.7%) and female (−14.7%) mice. Several diastolic function parameters were negatively regulated in males and females such as E/A, E/E′, isovolumetric relaxation time. Global longitudinal (GLS) and circumferential (GCS) strains were reduced by Iso in both sexes, GLS by 31% and GCS by about 20%. For the segmental LV analysis, we measured strain, strain rate, reverse strain rate, peak speckle displacement and peak speckle velocity in the parasternal long axis. We observed that radial strain of the LV posterior segments were more severely modulated by Iso than those of the anterior wall in males. In females, on the other hand, both posterior and anterior wall segments were negatively impacted by Iso. Longitudinal strain showed similar results to the radial strain for both sexes. Strain rate, on the other hand, was only moderately changed by Iso. Reverse strain rate measurements (an index of diastolic function) showed that posterior LV segments were negatively regulated by Iso. We then studied the animals 5 and 17 weeks after Iso treatment. Compared to control mice, LV dilation was still present in males. Ejection fraction was decreased in mice of both sex compared to control animals. Diastolic function parameters, on the other hand, were back to normal. Taken together, our study indicates that segmental strain analysis can identify LV regions that are more negatively affected by a cardiotoxic agent such as Iso. In addition, cessation of Iso was not accompanied with a complete restoration of cardiac function after four months.

228 Several factors can limit acquisition of quality 4D echo scans in rodents as described before in several 229 studies [15,[19][20][21]. One is interference from anatomical structures (sternum, ribs and lungs) that often 230 obscure parts of the heart, making it difficult to visualize and to trace LV walls. Working with those low-231 quality 4D echo scans can significantly increase intra-observer variability and thus, reduce 232 reproducibility.
233 Despite these discrepancies between the methods used to evaluate EF, Iso increased both systolic LV 234 diameters and volumes in mice. A LV dilation was also apparent in 2D B-mode views, whereas the wall 235 thickness seemed to remain remained stable (see Figure 2, Videos S1 and S2). This LV remodeling helped 236 preserve cardiac output although a trend for a decrease was present. It is difficult to conclude that mice 237 were experiencing symptoms of HF, especially since cardiac output was mostly preserved. HF in small 238 rodents is often recognized by increased lungs weight after euthanasia or decreased exercise 239 performance [1]. We did not test resistance to forced exercise in our animals. 240 In the case of diastolic parameters measured by either pulse-wave or tissue Doppler, many of them 241 were negatively modulated by Iso in male mice. In females, only those measured by tissue Doppler were 242 affected by Iso and, to a lesser extent than for males. As in humans, defining diastolic dysfunction by 243 echo is difficult although clearly, Iso treatment caused diastolic function impairments, especially in male 244 mice.
245 Increased myocardial interstitial fibrosis is often related to diastolic dysfunction. Iso treatment has been 246 shown to induce intertitial fibrosis in mice in other studies. Collagen production was shown to by more 247 important in male mice receiving Iso than in females [14]. Among the other mechanisms that have been 248 proposed to explain HF induced by Iso is an increased rate of cardiomyocyte apoptosis [22]. Sur-249 activation of the beta-adrenergic system by Iso imposes an increased cardiac workload, which is 250 associated with increased consumption of oxygen by the myocardium. This can lead to increased 262 Using the strain analysis, we observed that the LV posterior wall was in general, more negatively 263 modulated by Iso than the anterior wall. The base and the mid-ventricle segments were the most 264 affected and the apex, to a lesser extent. This trend was clearer in males although it was also present in 265 females. Obviously, Iso treatment being longer and more severe in this study, triggered a compensatory 266 response [10]. LV dilatation had time to take place in males, which had to be accompanied by a 267 concomitant extracellular matrix remodeling. Fibrosis has been described in other studies looking at Iso 268 effects on the mouse myocardium [15,22]. This long exposure to Iso also likely brought a general down 269 regulation of beta-adrenergic receptors leaving non-receptor-mediated Iso effects play a significant part 270 here.
271 The reason for the posterior wall being more sensitive to Iso than the anterior one is not clear.
272 Differences in LV regional beta-receptor sub-types density have been reported before. Greater apical 273 beta-adrenergic receptor density or responsiveness has been described in humans, dogs, rats, cats and  Figure S2). This is associated with larger radial wall velocities for these 283 posterior LV segments. Here too, it was these posterior segments that were more negatively targeted by 284 Iso for both peak displacement and peak velocity. In the longitudinal direction, baseline values were 285 relatively similar to posterior and anterior segments. Manuscript to be reviewed 297 notice that again, the posterior wall segments in males are the ones with the more reduced reverse SR 298 suggesting LV stiffening caused by Iso seemed to mainly target this region.
299 STE strain analysis has been performed in the past in Iso-treated mice. In male mice receiving Iso for 300 either three or seven days, global radial and longitudinal strain and strain rate were reduced in PSLAX 301 and only strain rates were reduced in SAX. When concentrating on LV wall segments in PSLAX, the 302 authors found no regional differences suggesting that Iso effects on the LV after three or seven days 303 were mostly global [10]. Their dosage of Iso used was lower (5 vs. 30 mg/kg here) and duration of 304 treatment shorter, making it difficult to make comparison with our work. It is probable that in the 305 present study, more chronic mechanisms took place at the cellular and molecular levels, as mentioned 306 above. Interestingly, after only three days, An and collaborators observed in their mice, increased 307 myocardial fibrosis and hypertrophy. They did not mention if chamber dilatation was present but 308 reported LV wall thickening. We did not observe this in our mice after three weeks of Iso [10]. 309 We thus observe a clear reduction of global strain measurements in our animals and this was similar 310 between males and females. These parameters are highly sensitive to detect cardiac dysfunction but do 311 not provide regional information. Since most sex differences we observed were present at the regional 312 level, global longitudinal and circumferential strains were not informative.   p<0.0001 between Ctrl and Iso groups, respectively. ns: not significant.