Angiotensin II Overstimulation Leads to an Increased Susceptibility to Dilated Cardiomyopathy and Higher Mortality in Female Mice

Heart failure (HF) is associated with high mortality and affects men and women differently. The underlying mechanisms for these sex-related differences remain largely unexplored. Accordingly, using mice with cardiac-specific overexpression of the angiotensin II (ANGII) type 1 receptor (AT1R), we explored male-female differences in the manifestations of hypertrophy and HF. AT1R mice of both sexes feature electrical and Ca2+ handling alterations, systolic dysfunction, hypertrophy and develop HF. However, females had much higher mortality (21.0%) rate than males (5.5%). In females, AT1R stimulation leads to more pronounced eccentric hypertrophy (larger increase in LV mass/body weight ratio [+31%], in cell length [+27%], in LV internal end-diastolic [LVIDd, +34%] and systolic [LVIDs, +67%] diameter) and dilation (larger decrease in LV posterior wall thickness, +17%) than males. In addition, in female AT1R mice the cytosolic Ca2+ extrusion mechanisms were more severely compromised and were associated with a specific increased in Ca2+ sparks (by 187%) and evidence of SR Ca2+ leak. Altogether, these results suggest that female AT1R mice have more severe eccentric hypertrophy, dysfunction and compromised Ca2+ dynamics. These findings indicate that females are more susceptible to the adverse effects of AT1R stimulation than males favouring the development of HF and increased mortality.


Animals
Heterozygous male and female C57BL/6 AT1R (human angiotensin II type 1 receptor, AGTR1) mice aged of 6-8 months old (presence of ventricular hypertrophy) and their sexand age-matched wild-type littermate controls (CTL) were used. AT1R mice colony was followed from birth to 15 months of age and deaths were counted for the entire duration of the survival study. Generation of the AT1R mice with cardiac specific overexpression of the human AT1R has been previously published 1 . AGT1R was expressed under the control of the murine α-myosin heavy chain promoter, which is highly specific and directs transgene expression to the myocardium. The use of this promoter rules out the Heart Institute Animal Care Committee has also approved the animal procedures used in this study (reference number 2012-80-01).

Mouse ventricular myocyte isolation
Isolated mouse ventricular myocytes were obtained using previously published protocols 2,3 . Briefly, mice were administered heparin i.p. 15 minutes prior to anesthesia by isoflurane (2%) to avoid blood coagulation and were sacrificed by cervical dislocation.
Then, their heart was rapidly excised and hung on a modified Langendorff apparatus and retrogradely perfused at 37°C (2 mL/min Isolated ventricular myocytes were obtained by trituration and conserved at 4°C in KB until used on the same day.

Surface electrocardiogram (ECG)
ECG were performed as previously described 4,5 . Mice were anaesthetized with isoflurane (2%). Mouse body temperature was maintained at 37°C using a heating pad. Platinum electrodes were positioned subcutaneously and surface ECG was acquired in lead I configuration at the rate of 2 kHz using the Biopac acquisition System MP100 (EMKA Technologies, Paris, France). The signal was amplified, filtered at 100 Hz (low-pass) and 60 KHz (notch filter). Data were analyzed using ECG auto v2.8.1.18 (EMKA Technologies, Paris, France). Duration of QRS complex, QT interval and heart rate was measured by a blinded observer from signal averaged ECG recordings (500-1000 cardiac cycles). The QT intervals were corrected (QTc) for the heart rate using the adapted Bazett's formula in mice (QTc=QT/ (RR/100) 1/2 ). 6,7

Cellular electrophysiology
Voltage-clamp experiments were conducted using previously reported protocols 4 KHz). Series resistance were compensated at 75%. All currents were normalized to cell capacitance.

Echocardiography
Two-dimensional M-mode echocardiography was performed as described previously 4,13 .
Briefly, the day before echocardiography, the mice were anesthetized (2 % isoflurane, 1 L/min O2), and their anterior chest was shaved. The following day, echocardiography was  14 . LV mass to body weight ratio was then calculated.

Ca 2+ Transients
Experiments were performed accordingly to 9,15 .Ventricular myocytes were incubated 15 minutes in the dark with 10 μmol/L Fura-2AM (Molecular Probes were excluded from the analysis. A manual inspection was performed for each recording after the automated analysis to remove false positive.