Neuro-Glial Mechanisms Underlying Sex-Based Resilience to Cardiorespiratory Dysfunction in Heart Failure

Figure 1. Female rats present lower left ventricular hypertrophy induced by arteriovenous fistula. (a) Schematic representation of arteriovenous shunt to induce volume overload and a representative color Doppler ultrasound of an arteriovenous fistula (AVF). Note the arterial blood flow infiltrating into the cava vein. (b) Representative pseudo-color images of Doppler and pulse-wave Doppler showing blood flow velocities across the inferior vena cava and abdominal aorta of each experimental group. Note that CHF rats that underwent arteriovenous shunt showed arterial blood flowing into the venous circulation. (c) Representative M-mode echocardiography of left ventricle (LV) from one male Sham, one male CHF, one female Sham, and one female CHF rats. White arrows show LV end-diastolic diameter (LVEDD) and LV end-systolic diameter (LVESD). (d-g) Summary data of echocardiographic parameters: AVF flow, LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV), stroke volume (SV). Data were analyzed using One-way ANOVA and Holm–Sidak multiple comparisons test; *: P<0.05. n = 7–11 per group.

Figure 2. Female rats subjected to volume-overload CHF maintained LV systolic and diastolic function. (a) Right panel: Representative 20 consecutive intraventricular pressure–volume (PV) loops during vena cava occlusion to assess load-independent indexes of cardiac function. Left panel: Representative end-systolic and end-diastolic PV relationships (ESPVR and EDPVR), characterizing LV systolic and diastolic properties, respectively. Note that ESPVR is a regression between multiple correlated end-systolic volume (ESV) and end-systolic pressure (ESP) points, while EDPVR is non-linear and reflects LV compliance. (b-e) Summary data showing end-diastolic pressure (EDP) and end-systolic pressure (ESP), ESPVR, and EDPVR, respectively. Data were analyzed using One-way ANOVA and Holm–Sidak multiple comparisons test; *: P<0.05. n = 7–8 per group.

Figure 3. Female CHF rats display normal diastolic kinetic and bioavailability of cytosolic Ca22+ transients in cultured cardiomyocytes. (a) Representative line-scan recordings of electrically stimulated cytosolic Ca2+ transients in isolated cardiomyocytes of one male Sham, one male CHF, one female Sham, and one female CHF rats, and (b) power spectral density (PSD) from those representatives. Note that male CHF rats show impaired systolic Ca2+. (c-f) Summary data showing calcium removal rate, calcium dye fluorescence amplitude, time to peak, and diastolic calcium, respectively. Data were analyzed using One-way ANOVA and Holm–Sidak multiple comparisons test; *: P<0.05. Sham male, n = 4 (15 cells); CHF male, n = 3 (9 cells); Sham female, n = 3 (28 cells); CHF female, n = 4 (32 cells).

Figure 4. CHF induction in female rats failed to trigger cardiac autonomic imbalance and arrhythmias. (a) Representative traces of resting blood pressure–first derivative (dP/dt, upper panel) and heart rate (HR, bottom panel) in one male Sham, one male CHF, one female Sham, and one female CHF rat. Arrowheads show ventricular arrhythmic events. (b) Representative HR traces following acute administration of propranolol (1 mg/kg i.p., upper panel) and acute administration of atropine (1 mg/kg i.p., bottom panel) to evaluate cardiac sympathetic tone and parasympathetic tone, respectively. Note that the male CHF rat showed a larger response to propranolol and a decreased response to atropine compared with the female CHF rat. (c,d) Summary data showing resting basal HR and arrhythmia incidence, respectively. (e,f) Summary data showing the chronotropic response as ΔHR to propranolol and atropine, respectively. Data were analyzed using One-way ANOVA and Holm–Sidak multiple comparisons test; *: P<0.05. n = 5–6 per group.

Figure 5. Male CHF rats but not female CHF rats displayed RVLM chronic neuronal activation and remodeling of astrocyte complexity. (a) Representatives immunoblot against FosB transcription factor and quantification of FosB and FosB2 protein expression of RVLM micropunches. (b) Representative confocal images showing GFAP+ astrocytes in male and female Sham and CHF rats. In sets: higher magnification with GFAP (red), TH (green), and DAPI (blue). Scale bar = 100 μm (c) 3D reconstructions and skeletal representations of single GFAP+ astrocytes per group, with convex hull and Sholl topology visualization. (d-e) Summary data showing quantification of astrocyte cable length, number of branches, and convex hull boundary size, respectively. (g) Number of intersections as a function of the distance from the nucleus. (h) Pie charts showing the proportion of astrocytes in each morphological complexity category (low, medium, high) derived from unsupervised clustering on PCA-reduced morphometric data. Data were analyzed using One-way ANOVA and Holm–Sidak multiple comparisons test; *: P<0.05. n = 80–100 cells per group; n = 3–4 animals per group.

Figure 6. Female CHF rats showed no signs of disordered breathing at rest. (a) Representative ventilatory flow recordings (top) and frequency (bottom) obtained from one male Sham, one male CHF, one female Sham, and one female CHF rat. (b) Representative 3D Poincaré plots displaying breath-to-breath interval variability of one rat per group. (c) Frequency histograms showing the distribution of tidal volume (VT) cycle amplitudes. (d,e) Summary data showing short-term (SD1) and long-term (SD2) breathing interval variability. (f,g) Summary data showing the coefficient of variation of VT and irregularity score (IS). Data were analyzed using One-way ANOVA and Holm–Sidak multiple comparisons test; *: P<0.05. n = 6–10 per group.

Figure 7. Central chemoreflex drive remains unchanged in female CHF animals. (a) Representative ventilatory traces after hypercapnia challenge (FiCO2 7%) from one animal per group. (b-d) Summary data showing the magnitude of response in terms of tidal volume (VT), respiratory frequency (RF), and the hypercapnic ventilatory response (HCVR), respectively. Data were analyzed using One-way ANOVA and Holm–Sidak multiple comparisons test; *: P<0.05. n = 6–10 per group.