A Primer on Incorporating Sex as a Biological Variable into the Conduct and Reporting of Basic and Clinical Research Studies

The recent move to require sex as a biological variable (SABV), which includes gender, into the reporting of research published by the American Journal of Physiology - Heart and Circulatory Physiology follows a growing, and much-needed, trend by journals. Understandably, there is concern over how to do this without adding considerable work, especially if one's primary research focus is not on elucidating sex/gender differences. The purpose of this article is to provide additional guidance and examples on how to incorporate SABV into the conduct and reporting of basic and clinical research. Using examples from our research, which includes both studies focused and not focused on sex/gender differences, we offer suggestions for how to incorporate SABV into basic and clinical research studies.

Comprehensive analyses of the inotropic compound omecamtiv mecarbil in rat and human cardiac preparations

Omecamtiv mecarbil (OM), a myosin activator, was reported to induce complex concentration- and species-dependent effects on contractile function and clinical studies indicated a low therapeutic index with diastolic dysfunction at concentrations above 1 µM. To further characterize effects of OM in a human context and under different preload conditions, we constructed a setup that allows isometric contractility analyses of human induced pluripotent stem cell (hiPSC)-derived engineered heart tissues (EHTs). The results were compared to effects of OM on the very same EHTs measured under auxotonic conditions. OM induced a sustained, concentration-dependent increase in time-to-peak under all conditions (maximally 2-3 fold). Peak force, in contrast, was increased by OM only in human, but not rat EHTs and only under isometric conditions, varied between hiPSC lines and showed a biphasic concentration-dependency with maximal effects at 1 µM. Relaxation time tended to fall under auxotonic and strongly increase under isometric conditions, again with biphasic concentration-dependency. Diastolic tension concentration-dependently increased under all conditions. The latter was reduced by an inhibitor of the mitochondrial sodium calcium exchanger (CGP-37157). OM induced increases in mitochondrial oxidation in isolated cardiomyocytes, indicating that OM, an inotrope that does not increase intracellular and mitochondrial Ca2+, can induce mismatch between an increase in ATP and ROS production and unstimulated mitochondrial redox capacity. Taken together, we developed a novel setup well suitable for isometric measurements of EHTs. The effects of OM on contractility and diastolic tension are complex with concentration-, time-, species- and loading-dependent differences. Effects on mitochondrial function require further studies.

Quantification of Murine Myocardial Infarct Size using 2D and 4D High Frequency Ultrasound

Background: Ischemic heart disease is the leading cause of death in the United States, Canada, and worldwide. Severe disease is characterized by coronary artery occlusion, loss of blood flow to the myocardium, and necrosis of tissue, with subsequent remodeling of the heart wall, including fibrotic scarring. The current study aims to demonstrate the efficacy of quantitating infarct size via 2D echocardiographic akinetic length and 4D echocardiographic infarct volume and surface area as in vivo analysis techniques. We further describe and evaluate a new surface area strain analysis technique for estimating myocardial infarction (MI) size after ischemic injury. Methods: Experimental MI was induced in mice via left coronary artery ligation. Ejection fraction and infarct size were measured through 2D and 4D echocardiography. Infarct size established via histology was compared to ultrasound-based metrics via linear regression analysis. Results: 2D echocardiographic akinetic length (r = 0.76, p = 0.03), 4D echocardiographic infarct volume (r = 0.85, p = 0.008) and surface area (r = 0.90, p = 0.002) correlate well with histology. While both 2D and 4D echocardiography were reliable measurement techniques to assess infarct, 4D analysis is superior in assessing asymmetry of the left ventricle and the infarct. Strain analysis performed on 4D data also provides additional infarct sizing techniques, which correlate with histology (surface strain: r = 0.94, p < 0.001, transmural thickness: r = 0.76, p = 0.001). Conclusions: 2D echocardiographic akinetic length, 4D echocardiography ultrasound and strain provide effective in vivo methods for measuring fibrotic scarring after MI.

Sex differences in rat renal arterial responses following exercise training

During aerobic exercise, hemodynamic alterations occure; while blood flow in skeletal muscle arteries increases, it decreases in visceral vessels due to mesenterial vasoconstriction. However, maintaining renal blood flow during intensive sport is also a priority. Our aim was to investigate the changes of vascular reactivity and histology of isolated renal artery of male and female rats in response to swim-training. Wistar rats were distributed into four groups: male sedentary (MSed), male trained (MTr), female sedentary (FSed), and female trained (FTr). Trained animals underwent a 12-week-long intensive swimming program. Vascular function of isolated renal artery segments was examined by wire myography. Phenylephrine-induced contraction was lower in FSed compared to MSed animals, and it was decreased by training in male but not in female animals. Inhibition of cyclooxygenases by indomethacin reduced contraction in both sedentary groups, and in MTr but not in FTr animals. Inhibition of nitric oxide production increased contraction in both trained groups. Acetylcholine induced relaxation was similar in all experimental groups showing predominant NO-dependency. Elastin and smooth muscle cell actin density was reduced in female rats after aerobic training. This study shows that, as a result of 12-weeks-long training, there are sex differences in renal arterial responses following exercise training. Swimming moderates renal artery vasoconstriction in male animals, while it depresses elastic fiber and smooth muscle actin density in females.

Sympathetic neural responses to sleep disorders and insufficiencies

Short sleep duration and poor sleep quality are associated with cardiovascular risk, and sympathetic nervous system (SNS) dysfunction appears to be a key contributor. The present review will characterize sympathetic function across several sleep disorders and insufficiencies in humans, including sleep deprivation, insomnia, narcolepsy, and obstructive sleep apnea (OSA). We will focus on direct assessments of sympathetic activation (e.g., plasma norepinephrine and muscle sympathetic nerve activity), but include heart rate variability (HRV) when direct assessments are lacking. The review also emphasizes sex as a key biological variable. Experimental models of total sleep deprivation and sleep restriction are converging to support epidemiological studies reporting an association between short sleep duration and hypertension, especially in women. A systemic increase of SNS activity via plasma norepinephrine is present with insomnia, and has also been confirmed with direct, regionally-specific evidence from microneurographic studies. Narcolepsy is characterized by autonomic dysfunction via both HRV and microneurographic studies, but with opposing conclusions regarding SNS activation. Robust sympathoexcitation is well documented in OSA, and is related to baroreflex and chemoreflex dysfunction. Treatment of OSA with continuous positive airway pressure results in sympathoinhibition. In summary, sleep disorders and insufficiencies are often characterized by sympathoexcitation and/or sympathetic/baroreflex dysfunction, with several studies suggesting women may be at heightened risk.

Is 'Not Different' Enough to Conclude Similar Cardiovascular Responses Across Sexes?

The number of research studies investigating whether similar or different cardiovascular responses or adaptations exist between males and females are increasing. Traditionally, difference-based statistical methods (e.g., t-test, ANOVA, etc.) have been implemented to compare cardiovascular function between males and females, with a P-value >0.05 used to denote similarity between sexes. However, an absence of evidence (i.e., large P-value) is not evidence of absence (i.e., no sex differences). Equivalence testing determines whether two measures or groups provide statistically equivalent outcomes, in that they differ by less than an 'ideally prespecified' smallest effect size of interest. Our perspective discusses the applicability and utility of integrating equivalence testing when conducting sex comparisons in cardiovascular research. An emphasis is placed on how cardiovascular researchers may conduct equivalence testing across multiple study designs (e.g., cross-sectional comparisons, repeated measures intervention, etc.). The strengths and weaknesses of this statistical tool are discussed. Equivalence analyses are relatively simple to conduct, may be used in conjunction with traditional hypothesis testing to interpret findings, and permits the determination of statistically equivalent responses between sexes. We recommend that cardiovascular researchers consider implementing equivalence testing to better our understanding of similar and different cardiovascular processes between sexes.

Microcirculatory and glycocalyx properties are lowered by high salt diet but augmented by western diet in genetically heterogeneous mice

Many individuals in industrialized societies consume a high salt, western diet, however, the effects of this diet on microcirculatory properties and glycocalyx barrier function are unknown. Young genetically heterogeneous male and female mice underwent 12 weeks of normal chow diet (NC), NC diet with 4% salt (NC4%), western diet (WD), or WD with 4% salt (WD4%). Microcirculatory properties and glycocalyx barrier function were evaluated in the mesenteric microcirculation using an intravital microscope equipped with an automated capture and analysis system. Total microvascular density summed across 4-25 μm microvessel segment diameters was lower in NC4% compared to NC and WD (P<0.05). Perfused boundary region (PBR), a marker of glycocalyx barrier function, averaged across 4-25 μm microvessel segment diameters was similar between NC and NC4%, as well as between WD and WD4% (P>0.05). PBR was lower in WD and WD4% compared to NC and NC4% (P<0.05), indicating augmented glycocalyx barrier function in WD and WD4%. There were strong, inverse relationships between PBR and adiposity and blood glucose (r=-0.44 to -0.61, P<0.05). In summary, NC4% induces deleterious effects on microvascular density, whereas WD augments glycocalyx barrier function. Interestingly, the combination of high salt, western diet in WD4% resulted in lower total microvascular density like NC4% and augmented glycocalyx barrier function like WD. These data suggest distinct microcirculatory adaptations to high salt and western diets that coincide when these diets are combined in young genetically heterogeneous male and female mice.

Nitric Oxide-mediated Cutaneous Microvascular Function is not Altered in Young Adults Following Mild-to-Moderate SARS CoV-2 Infection.

Vascular dysfunction has been reported in adults who have recovered from COVID-19. To date, no studies have investigated the underlying mechanisms of persistent COVID-19-associated vascular dysfunction. PURPOSE: To quantify nitric oxide (NO)-mediated vasodilation in healthy adults who have recovered from SARS-CoV-2 infection. We hypothesized that COVID-19-recovered adults would have impaired NO-mediated vasodilation compared to adults who have not had COVID-19. METHODS: We performed a cross-sectional study including: 10 (5M/5W, 24 ± 4yrs) healthy control (HC) adults who were unvaccinated for COVID-19, 11 (4M/7W, 25 ± 6yrs) healthy vaccinated (HV) adults, and 12 (5M/7W, 22 ± 3yrs) post-COVID-19 (PC, 19 ± 14wks) adults. COVID-19 symptoms severity (survey) were assessed. A standardized 39°C local heating protocol was used to assess NO-dependent vasodilation via perfusion (intradermal microdialysis) of 15 mM NG-nitro-l-arginine methyl ester during the plateau of the heating response. Red blood cell flux was measured (laser-Doppler flowmetry) and cutaneous vascular conductance (CVC = flux/mmHg) was expressed as a percentage of maximum (28mM sodium nitroprusside + 43°C). RESULTS: The local heating plateau (HC: 61 ± 20%, HV: 60 ± 19%, PC: 67 ± 19%, p=0.80) and NO-dependent vasodilation (HC: 77 ± 9%, HV: 71 ± 7%, PC: 70 ± 10%, p=0.36) were not different among groups. Neither symptom severity (25 ± 12 AU) nor time since diagnosis correlated with the NO-dependent vasodilation (r=0.46, p=0.13; r=0.41, p=0.19, respectively). CONCLUSION: Healthy adults who have had mild-to-moderate COVID-19 do not have altered NO-mediated cutaneous microvascular function.

Assessing hemodynamics from the photoplethysmogram to gain insights into vascular age: A review from VascAgeNet

The photoplethysmogram (PPG) signal is widely measured by clinical and consumer devices, and it is emerging as a potential tool for assessing vascular age. The shape and timing of the PPG pulse wave are both influenced by normal vascular ageing, changes in arterial stiffness and blood pressure, and atherosclerosis. This review summarises research into assessing vascular age from the PPG. Three categories of approaches are described: (i) those which use a single PPG signal (based on pulse wave analysis); (ii) those which use multiple PPG signals (such as pulse transit time measurement); and (iii) those which use PPG and other signals (such as pulse arrival time measurement). Evidence is then presented on the performance, repeatability and reproducibility, and clinical utility of PPG-derived parameters of vascular age. Finally, the review outlines key directions for future research to realise the full potential of photoplethysmography for assessing vascular age.

Analysis of Drosophila cardiac hypertrophy by micro-computerized tomography for genetic dissection of heart growth mechanisms

Heart failure is often preceded by pathological cardiac hypertrophy, a thickening of the heart musculature driven by complex gene regulatory and signaling processes. The Drosophila heart has great potential as a genetic model for deciphering the underlying mechanisms of cardiac hypertrophy. However, current methods for evaluating hypertrophy of the Drosophila heart are laborious and difficult to carry out reproducibly. Here we demonstrate that micro-computerized tomography (microCT) is an accessible, highly reproducible method for non-destructive, quantitative analysis of Drosophila heart morphology and size. To validate our microCT approach for analyzing Drosophila cardiac hypertrophy, we show that expression of constitutively active Ras (Ras85DV12), previously shown to cause hypertrophy of the fly heart, results in significant thickening of both adult and larval heart walls when measured from microCT images. We then show using microCT analysis that genetic upregulation of store-operated Ca2+ entry (SOCE) driven by expression of constitutively active Stim (StimCA) or Orai (OraiCA) proteins also results in significant hypertrophy of the Drosophila heart, through a process that specifically depends on Orai Ca2+ influx channels. Intravital imaging of heart contractility revealed significantly reduced end diastolic and systolic dimensions in StimCA and OraiCA expressing hearts, consistent with the hypertrophic phenotype. These results demonstrate that increased SOCE activity is an important driver of hypertrophic cardiomyocyte growth, and demonstrate how microCT analysis combined with tractable genetic tools in Drosophila can be used to delineate molecular signaling processes that underlie cardiac hypertrophy and heart failure.