Occlusion preconditioned mice are resilient to hypobaric hypoxia-induced myocarditis and arrhythmias due to enhanced immunomodulation, metabolic homeostasis, and antioxidants defense

Abstract

Background: Sea-level residents experience altitude sickness when they hike or visit altitudes above ~2,500 m due to the hypobaric hypoxia (HH) conditions at such places. HH has been shown to drive cardiac inflammation in both ventricles by inducing maladaptive metabolic reprogramming of macrophages, which evokes aggravated proinflammatory responses, promoting myocarditis, fibrotic remodeling, arrhythmias, heart failure, and sudden deaths. The use of salidroside or altitude preconditioning (AP) before visiting high altitudes has been extensively shown to exert cardioprotective effects. Even so, both therapeutic interventions have geographical limitations and/or are inaccessible/unavailable to the majority of the population as drawbacks. Meanwhile, occlusion preconditioning (OP) has been extensively demonstrated to prevent hypoxia-induced cardiomyocyte damage by triggering endogenous cardioprotective cascades to mitigate myocardial damage. Herein, with the notion that OP can be conveniently applied anywhere, we sought to explore it as an alternative therapeutic intervention for preventing HH-induced myocarditis, remodeling, and arrhythmias.

Methods: OP intervention (6 cycles of 5 min occlusion with 200 mmHg for 5 min and 5 min reperfusion at 0 mmHg - applying to alternate hindlimb daily for 7 consecutive days) was performed, and its impact on cardiac electric activity, immunoregulation, myocardial remodeling, metabolic homeostasis, oxidative stress responses, and behavioral outcomes were assessed before and after exposure to HH in mice. In humans, before and after the application of OP intervention (6 cycles of 5 min occlusion with 130% of systolic pressure and 5 min reperfusion at 0 mmHg - applying to alternate upper limb daily for 6 consecutive days), all subjects were assessed by cardiopulmonary exercise testing (CPET).

Results: Comparing the outcomes of OP to AP intervention, we observed that similar to the latter, OP preserved cardiac electric activity, mitigated maladaptive myocardial remodeling, induced adaptive immunomodulation and metabolic homeostasis in the heart, enhanced antioxidant defenses, and conferred resistance against HH-induce anxiety-related behavior. Additionally, OP enhanced respiratory and oxygen-carrying capacity, metabolic homeostasis, and endurance in humans.

Conclusions: Overall, these findings demonstrate that OP is a potent alternative therapeutic intervention for preventing hypoxia-induced myocarditis, cardiac remodeling, arrhythmias, and cardiometabolic disorders and could potentially ameliorate the progression of other inflammatory, metabolic, and oxidative stress-related diseases.

Keywords: antioxidant responses; arrhythmias; hypobaric hypoxia; immunomodulation; metabolic homeostasis; myocardial remodeling; myocarditis; remote ischemic preconditioning.

Figure 1

OP preserves cardiac electric activity during hypobaric hypoxia. (A) Representative electrocardiography (EKG) of Normobaric Normoxia (NN), Hypobaric Hypoxia (HH), Altitude Preconditioned (AP), Altitude Preconditioned before HH exposure (APHH), Occlusion Preconditioned (OP) and Occlusion Preconditioned before HH exposure (OPHH) mice. P wave: atrial depolarization; Q wave: Interventricular septum depolarization; R wave: Ventricular depolarization; S wave: Purkinje fibres depolarization; J wave: Early ventricular repolarization; T wave: End of ventricular repolarization. (B-G) Graphical presentation of EKG parameters including; Heart Rate (HR), QT Interval, corrected QT Interval (QTc), JT Interval, Tpeak Tend Interval, and ST Height. (n= 5-9 mice per experimental group). ^$p<0.05, ^$$p<0.01, ^$$$p<0.001 HH vs NN; ^&p<0.05 APHH vs AP; ^#p<0.05 OPHH vs OP; *p<0.05, **p<0.01. Data are expressed as mean ± SEM. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc analysis.

Figure 2

OP mitigates myocardial hypertrophy, injury, and fibrosis during hypobaric hypoxia. (A) Graphical representation of Body Weight (BW) trends of 14 days period by Normobaric Normoxia (NN), Hypobaric Hypoxia (HH), Altitude Preconditioned (AP), Altitude Preconditioned before HH exposure (APHH), Occlusion Preconditioned (OP) and Occlusion Preconditioned before HH exposure (OPHH) mice (n= 7-15 mice per experimental group). (B) Graphical presentation of Heart Weight (HW)/BW ratio (n= 5-10 mice per experimental group). (C-H) Indexes for cardiac hypertrophy assessment, including; Representative Wheat germ agglutinin (WGA) staining and Graphical presentation of Cardiomyocyte surface area (n=8-12 cells per section per 4-6 heart per group), Atrial natriuretic peptide (ANP) and Brain natriuretic peptide (BNP) concentrations (n=6-8 hearts per group), Representative Immunoblotting of GATA4 and its Graph plot (n= 3 hearts per group). ELISA were performed in triplicates. Immunoblots were performed in triplicates, and each blot band in the representative blot is an independent biological sample. (I, J) Representative Masson’s trichome staining and Graphical presentation of collagen volume fraction (CVF) showing the extent of fibrosis among experimental groups (n= 3-6 sections per 4,5 hearts per group). ^$p<0.05, ^$$$p<0.001 HH vs NN; ^&p<0.05, ^&&&p<0.001 APHH vs AP; ^##p<0.01, ^###p<0.001 OPHH vs OP; ^*p<0.05, ^**p<0.01, ^***p<0.001. Data are expressed as mean ± SEM. Data were analyzed using one-way anova, followed by Tukey’s post hoc analysis.

Figure 3

OP induces adaptive immunomodulation and metabolic homeostasis during hypobaric hypoxia. (A, B) Representative flow cytometry of myocardial macrophages gated on CD45⁺CD11b⁺F4/80 and Graphical plots of CD86⁺ and CD206⁺. Normobaric Normoxia (NN), Hypobaric Hypoxia (HH), Altitude Preconditioned (AP), Altitude Preconditioned before HH exposure (APHH), Occlusion Preconditioned (OP), and Occlusion Preconditioned before HH exposure (OPHH) mice hearts (n=4 hearts per group). ^$p<0.05 vs NN_CD86+; ^##p<0.01, ^###p<0.001 vs HH_CD206+ (C) Graphical presentation of sera cardiac troponin I (cTnI) concentrations. (D, E) Inflammatory mediators; Inducible nitric oxide synthase (iNOS) and Arginase-1 (Arg-1) concentrations assessed by ELISA using myocardia lysates. (F-I) Inflammatory cytokines; Interleukin (IL)-1β, IL-18, IL-10, and transforming growth factor (TGF)-β concentrations assessed by ELISA using myocardia lysates. All ELISA were performed in triplicates (n= 5-8 mice per group). (J-L) Representative Oil Red O (ORO) and Periodic Acid Schiff (PAS) staining of myocardial sections and their respective graphical presentations showing lipid and glycogen depositions percentages (n=4-6 sections per 4-6 mice per group). Yellow outlined boxes are original myocardial portions and red outline boxes are their zoomed-in (5x) inserts to show positive stained area (indicated with black arrows). (M-O) Representative Immunoblotting of Scarb3 and Slc2a1 and their respective Graphical plots; each blot band in the representative blot is an independent biological sample (n= 3 hearts per group). ^$$p<0.01, ^$$$p<0.001 HH vs NN; ^&&p<0.01 APHH vs AP; ^*p<0.05, ^**p<0.01, ^***p<0.001. Data are expressed as mean ± SEM. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc analysis.

Figure 4

OP induces adaptive modulation of oxidative stress responses. (A-D) Representative Immunoblotting of Hypoxia-inducible factors (HIF)-1α, HIF-2α, and nuclear factor erythroid 2–related factor 2 (Nrf2), and their respective Graphical plots; each blot band in the representative blot is an independent biological sample (n= 4 hearts per group). Normobaric Normoxia (NN), Hypobaric Hypoxia (HH), Altitude Preconditioned (AP), Altitude Preconditioned before HH exposure (APHH), Occlusion Preconditioned (OP), and Occlusion Preconditioned before HH exposure (OPHH) mice hearts. (E-H) Antioxidants state indexes; Graphical plots of the concentrations of redox cofactors, Nicotinamide adenine dinucleotide (NAD)+hydrogen (NADH) and their ratio, as well as the Total antioxidant capacity (T-AOC) of the myocardia (n=8 hearts per group). (I) Graphical plot of survival data in Kaplan-Meier estimator (n=18 mice per group). ^$p<0.05, ^$$p<0.01, ^$$$p<0.001 HH vs NN; ^&p<0.05 APHH vs AP; ^#p<0.05 OPHH vs OP; ^**p<0.01, ^***p<0.001. Data are expressed as mean ± SEM. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc analysis. Survival curves were analyzed with the Kaplan-Meier estimator.

Figure 5

OPHH mice are resilient to HH-associated maladaptive behavioral outcomes. (A-H) Locomotive and exploratory behavioral assessment indexes from Open field test (OFT), including; Representative plots of Total distance moved and its graphical presentation, Representative plots of Average velocity, Mobile duration, and Immobile duration, and their graphical presentations and Representative heat-maps of Relative zone entries and its graphical presentation (n=6-10 mice per group). Normobaric Normoxia (NN), Hypobaric Hypoxia (HH), Altitude Preconditioned (AP), Altitude Preconditioned before HH exposure (APHH), Occlusion Preconditioned (OP), and Occlusion Preconditioned before HH exposure (OPHH) mice. (I, J) Anxiety-related and exploratory behavioral assessment indexes from Elevated plus maze (EPM), including; Representative heat-maps of Relative arm entries and its graphical presentation (n=6-10 mice per group). ^$p<0.05 HH vs NN; *p<0.05. Data are expressed as mean ± SEM. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc analysis.

Figure 6

OP enhances respiratory and oxygen-carrying capacity in humans. (A-M) Cardiopulmonary Exercise Test (CPET) indexes for respiratory and oxygen carrying capacity in humans (n=14 human volunteers), Before occlusion preconditioning (BOP) and After occlusion preconditioning (AOP), including; Graphical plots of Heart Rates (HR), Systolic blood pressures (Psys), Diastolic blood pressures (Pdia), Vital capacity of inhalation (IN), Vital capacity of exhalation(EX), Expiratory reserve volume, Minute ventilation (V’E), Carbon dioxide output (V’CO₂), Oxygen uptake (V’O₂), Oxygen saturation (SpO₂), Oxygen pulse (V’O₂/HR), Metabolic equivalent of task (MET) and Respiratory exchange ratio (RER). Rest, Warm-up, Second ventilation threshold (VT2), Maximal workload (Max Watts) and Recovery are timepoints of interest during the CPET. *p<0.05, **p<0.01 AOP vs BOP. Data are expressed as mean ± SEM. Data were analyzed using an unpaired t-test for comparing two groups and two-way ANOVA for grouped analysis.

Figure 7

β₂AR is implicated in OP-induced adaptive responses against hypobaric hypoxia (A) Graphical presentation of Body weight (BW) alteration trends among Wild type (Adrb2^+/+) and β₂AR knockout (Adrb2^-/-) mice in experiment groups; Normobaric Normoxia (NN), Hypobaric Hypoxia (HH), Altitude Preconditioned (AP), Altitude Preconditioned before HH exposure (APHH), Occlusion Preconditioned (OP) and Occlusion Preconditioned before HH exposure (OPHH) (n=5-15 mice per group). (B-D) Graphical presentation of electrocardiogram (EKG) indexes, including; Heart Rate, QT Interval, and JT interval (n=5-9 mice per group). (E-G) Inflammatory biomarker; C-reactive protein, Inducible nitric oxide synthase (iNOS), and Arginase-1 (Arg-1) concentrations assessed by ELISA. Assays were performed in triplicates (n=4 mice per group). (H-K) Representative Immunoblotting of Hypoxia-inducible factors (HIF)-1α, HIF-2α, and nuclear factor erythroid 2–related factor 2 (Nrf2), and their respective Graphical plots; each blot band in the representative blot is an independent biological sample (n= 3 hearts per group). (L) Graphical plots of the concentrations of Total antioxidant capacity (T-AOC) of myocardia (n=4 hearts per group).^$p<0.05, ^$$p<0.01, ^$$$p<0.001 vs NN_Adrb2+/+; ^&p<0.05, ^&&p<0.01, ^&&&p<0.001 vs HH_Adrb2+/+; ^##p<0.01 vs OP_Adrb2+/+; ^¥p<0.05, ^¥¥p<0.01, ^¥¥¥p<0.001 vs OPHH_Adrb2+/+;^!!!p<0.001 vs NN_Adrb2-/-; ^%p<0.001 vs HH_Adrb2-/-; ^£p<0.05, ^££p<0.01, ^£££p<0.001 vs OP_Adrb2-/-. Data are expressed as mean ± SEM. Data were analyzed using two-way ANOVA.