Oxygen supplementation, though an indispensable mechanism of treatment in the ICU, negatively impacts cardiovascular parameters and increases mortality rates compared to normoxia treatment [24, 25]. A retrospective cohort study of ninety-seven ICU facilities found that the percentage of patients receiving supplemental oxygen therapy at any hour was 39.5% (± 15.2%) , indicating that many ICU patients will receive oxygen supplementation at some point and, in turn, become exposed to hyperoxic conditions. Moreover, the use of emergent supplemental oxygen is increasing on a global scale, as it is the most commonly used treatment for COVID-19 patients . To reiterate, men and women exhibit inherent differences in respiratory function. Moreover, those that present most frequently to the ICU are individuals suffering from existing comorbidities, and one of the most common of such conditions is diabetes mellitus. Thus, it is imperative to understand how cardiac remodeling is influenced by hyperoxia, T1DM, and sex.
Distinct features of Akita males with regards to physical parameters
In line with our previous findings [14, 26, 27], we found all mice exposed to hyperoxic conditions, irrespective of gender or strain, experienced a significant decrease in body weight compared to normoxia controls (Fig. 1A). We also observed that Akita male mice at both baseline normoxia and hyperoxia experienced a significant (p < 0.0005) decrease in body weight compared to WT male mice exposed to either treatment (Fig. 1A).
With respect to heart weight, we have previously established that hyperoxia increases heart weights in WT males, while heart weights of WT females decrease [14, 22, 23]. Interestingly, we found that Akita males experience a decrease in heart weight after hyperoxia, similar to what is observed in hyperoxic female mice (both Akita and WT) (Fig. 1B). Moreover, Akita LV, RV, septum, and pooled cardiomyocyte areas significantly decreased after hyperoxia (with the exception of Akita male RV cardiomyocytes, which did decrease albeit not in a statistically significant manner) (Fig. 1E to I).
Regarding hormonal fluctuations, Akita males exhibit significantly increased (p<0.05) serum estradiol levels compared to Akita females (Fig. 5). Moreover, while studies have reported significantly decreased androgen levels in Akita males [28, 29], we are the first to report increased estradiol levels in male Akita mice compared to their female counterparts at both baseline normoxia and hyperoxia conditions.
Concerning the small size of Akita male mice, the T1DM genotype may be influential. In the clinical model, T1DM is associated with sub-optimal growth due to microvascular complications such as nephropathy . Additionally, the decreased serum levels of testosterone which have been previously observed in Akita males also likely contributes to their smaller size (44, 45). Testosterone has an integral role in glucose-stimulated insulin secretion (GSIS) in men , and recent reports indicate testosterone deficiency is common in men with both T1DM and T2DM [31-33]. Thus, testosterone deficiency as a consequence of the T1DM genotype may lead to impaired insulin secretion, thereby making weight gain more difficult.
The T1DM genotype may also contribute to the observation that hyperoxic Akita male mice did not display cardiac hypertrophy but instead experienced reductions in heart size similar to female mice. In cardiomyocytes, testosterone induces hypertrophy through both activation of the rapamycin complex 1 (mTORC1) pathway and glucose uptake facilitated by AMP-activated protein kinase (AMPK) activation . Moreover, androgen receptors themselves can alter the cardiac phenotype by modulating hypertrophy in cardiac myocytes [35, 36]. Taken together, these factors may help explain why Akita male mice do not experience hyperoxia-induced cardiac hypertrophy, which was otherwise evident in wild-type male mice as we reported previously [14, 23, 37].
Finally, statistical analysis determined genotype to have the most significant effect on physical parameters. Importantly, we also observed a positive correlation between heart and body weights, but a negative correlation between heart rates and QRS and/or JT intervals (Supplemental material). Studies have shown that in malnourished adults, body and heart weight decrease in tandem, while the resultant cardiac hypotrophy is associated with a prolonged QTc interval [38, 39], as observed in this experiment. Thus, the T1DM genotype has an important role in influencing physical parameters in Akita male mice, which we believe influence electrophysiological outcomes. Moreover, this influence is evident at both baseline normoxia and hyperoxia.
Akita male mice demonstrate distinct modulations in functional parameters at normoxia and hyperoxia
In the clinical setting, hyperoxic treatment results in modulations to functional parameters such as decreased cardiac output (CO) and stroke volume (SV) [40, 41]. Importantly, studies have shown T1DM patients may experience various functional parameter abnormalities even at normoxia, such as significantly lower %EF . Thus, hyperoxic treatment in T1DM patients may augment existing cardiac pathophysiology in this population.
Previously, we have reported that LVIDd, LVIDs, ESV, and EDV significantly decrease after hyperoxia in WT mice [14, 23]. Additionally, we also reported CO and SV in WT mice along with hyper-dynamic left ventricular ejection fraction (HDLVEF) [13, 14]. Our data from this experiment correspond well to these findings in that all mice, irrespective of gender or strain, experienced significant decreases in LVIDd, LVIDs, ESV, EDV, SV and CO (Table 1 & Fig. 2 D, E), in addition to a significant increase in %FS and %EF compared to normoxia counterparts (Fig. 2 B, C).
Akita male mice display distinct modulations in electrophysiological parameters
Abnormal electrocardiographic readings of T1DM patients are relatively common (about 35% at baseline) , increasing in number and severity over time . In this experiment, all experimental mice demonstrated bradycardia under hyperoxia, as demonstrated by the significant increases in RR intervals compared to normoxia controls (Fig 3B). While T1DM is not independently associated with bradycardia, such arrhythmias have been observed in hypoglycemic events , an important consideration for administration of supplemental oxygen to this patient population.
With regards to Akita mice specifically, atrioventricular conduction abnormalities were observed even at normal air and were further augmented by hyperoxic treatment, signified by the increased PR intervals (Fig. 3C). Importantly, the prolonged PR interval is associated with increased risk of atrial fibrillation (AF), as well as increased risk of mortality . Additionally, we observed ventricular depolarization abnormalities even at normal air in Akita mice, which were further augmented by hyperoxic treatment, as signified by the increased QRS intervals (Fig. 3D). Interestingly, Akita male mice also demonstrated elevated QTc intervals compared to female mice from either strain, indicating ventricular repolarization defects in Akita males. (Fig. 3E). Although it is widely accepted that females have higher QTc intervals than males, a prolonged QTc interval is a relatively common finding in T1DM patients overall [47, 48]. The prolongation of the QTc interval is independently associated with hypoglycemic attacks in T1DM patients and is an independent marker of mortality within this patient population [49, 50]. Therefore, male T1DM patients may be at greater risk for these adverse outcomes, and the hyperoxia-induced increase in QTc prolongation should therefore be considered as an additional risk factor when treating this subgroup. Moreover, the significantly higher (p<0.005) increase in serum LDH levels in Akita males after hyperoxia treatment compared to hyperoxic Akita femaless (Fig. 4) further elucidates such concerns.
Hyperoxia is the most significant risk factor of cardiac pathophysiology in Akita mice
To determine the mathematical impact of the risk factors studied (hyperoxia, gender, and T1DM genotype), Multi-Way Analysis of Variance (ANOVA) was performed. From our analysis, we determined physical parameters were influenced by all three risk factors, with genotype being the most statistically significant, followed by gender, and finally hyperoxia (Table 2). With regards to functional and electrophysiological parameters, hyperoxia had the greatest impact, followed by sex and genotype. Moreover, for almost all groups, correlation analysis indicated a positive correlation between body and heart weights, whereas a negative correlation was observed between heart rates and QRS and/or JT intervals (see Supplemental material), indicating possible influence of physical parameters on cardiac electrophysiology. Taken together, this data suggests that hyperoxic treatment has direct implications on cardiovascular function, which may augment inherent cardiac challenges experienced by T1DM patients.