Intrauterine growth restriction (IUGR) following prenatal hypoxia exposure leads to a higher risk of developing cardiovascular disease (CVD) in later life. Our aim was to evaluate cardiac susceptibility and its pathophysiological mechanisms following acute myocardial infarction (MI) in adult rat offspring exposed to prenatal hypoxia. Male and female rat offspring, which experienced normoxia (21% O2) or hypoxia (11% O2) in utero underwent sham or MI surgery at 12 weeks of age. Echocardiographic data revealed that both sexes had systolic dysfunction following MI surgery, independent of prenatal hypoxia. Male offspring exposed to prenatal hypoxia, however, had left ventricular dilatation, global dysfunction, and signs of diastolic dysfunction following MI surgery as evident by increased left ventricular internal diameter (LVID) during diastole (MI effect, P<0.01), Tei index (MI effect, P<0.001), and E/E′ ratio (prenatal hypoxia or MI effect, P<0.01). In contrast, diastolic dysfunction in female offspring was not as evident. Cardiac superoxide levels increased only in prenatal hypoxia exposed male offspring. Cardiac sarcoendoplasmic reticulum Ca2+-ATPase2a (SERCA2a) levels, a marker of cardiac injury and dysfunction, decreased in both male and female MI groups independent of prenatal hypoxia. Prenatal hypoxia increased cardiac ryanodine receptor 2 (RYR2) protein levels, while MI reduced RYR2 in only male offspring. In conclusion, male offspring exposed to prenatal hypoxia had an increased susceptibility to ischemic myocardial injury involving cardiac phenotypes similar to heart failure involving diastolic dysfunction in adult life compared with both offspring from healthy pregnancies and their female counterparts.

Introduction

Prenatal hypoxia, one of the most common outcomes of pregnancy complications, often leads to poor fetal growth resulting in intrauterine growth restriction (IUGR). IUGR, which accounts for 10–15% of all the pregnancies [1], has been associated with adverse long-term health outcomes in adult life including cardiovascular diseases (CVDs). It had been shown that offspring exposed to prenatal hypoxia exhibit permanent cardiac structural and physiological changes. For instance, fetal or neonatal hearts from IUGR animals have been shown to have fewer cardiomyocyte numbers [2], shorter sarcomere length [3], decreased cardiomyocyte proliferation [4], and increased apoptosis [5]. At molecular level, epigenetic modifications in prohypertensive and cardioprotective genes in the adrenal gland, brain tissues, and fetal hearts, have been reported in IUGR offspring from protein-restricted or hypoxic dams [68]. A suboptimal intrauterine environment, therefore, leads to adaptive changes at multisystem levels that may play a role in the manifestation of adult CVDs.

CVDs account for nearly one-third of all the deaths worldwide [9]. The major CVD is ischemic heart disease which is responsible for 38 and 46% of cardiovascular-related deaths in males and females, respectively [10]. Epidemiological and experimental studies have shown that populations born from hypoxic pregnancies are more susceptible to develop CVDs in later life in a sex-dependent manner (reviewed in [11,12]). Our laboratory as well as others have demonstrated that young adult rat offspring (3–4 months old) exposed to prenatal hypoxia are more susceptible to develop cardiac dysfunction after an episode of ex vivo ischemia/reperfusion (I/R) insult [13,14]. Interestingly, we did not observe in vivo cardiac dysfunction in young adult offspring exposed to prenatal hypoxia [15,16]; implying a subclinical phase of CVD manifestation in adult IUGR offspring. The manifestation of in vivo subclinical cardiovascular pathologies in offspring exposed to prenatal hypoxia becomes evident with exposure to secondary stressors such as aging [13] or a high-fat (HF) diet [17]. In particular, offspring exposed to prenatal hypoxia demonstrate a phenotype of in vivo cardiac diastolic dysfunction with normal systolic function [13,17].

It had been shown that oxidative stress plays a critical role in heart failure and correlates with the severity of heart failure [18]. Furthermore, several components involved in cardiac Ca2+ homeostasis, including sarcoendoplasmic reticulum Ca2+-ATPase2a (SERCA2a), have been shown to be altered in heart failure in studies in both the failing human heart and animal models of heart failure (reviewed in [19]). The pathophysiology of heart failure may involve multiple signaling pathways and is undoubtedly very complex and poorly understood; therefore, a better understanding of this condition might lead to prospective therapeutic strategies.

In adult IUGR rat offspring born to nutrient-restricted mothers, impaired Ca2+ handling due to alterations in sarcoendoplasmic reticulum properties [20], decreased ryanodine receptor 2 (RYR2) protein levels [20], and increased cardiac oxidative stress [2123] have been observed. These alterations in calcium handling might play a role in the increased susceptibility of IUGR offspring to adult CVDs; ultimately leading to heart failure in later life. In fact, the effect of prenatal hypoxia on long-term adverse cardiac function has previously been characterized by our lab [13,15] and others [14,24]. These characterizations have primarily been shown using the ex vivo I/R injury model, a condition often encountered in a clinical setting while treating ischemic heart disease. However, the cardiac susceptibility of adult offspring previously exposed to prenatal hypoxia to in vivo ischemic myocardial injury, is not known. Furthermore, our observations of signs of in vivo diastolic dysfunction in our animal models exposed to prenatal hypoxia [13] and when exposed to secondary stress factors [13,17] led us to examine cardiac function outcomes in offspring exposed to prenatal hypoxia following in vivo ischemic myocardial injury. We tested the hypothesis that there is an increase in vivo cardiac susceptibility of adult offspring exposed to prenatal hypoxia to ischemic myocardial injury using a myocardial infarction (MI) heart failure model. We also investigated the potential underlying molecular mechanisms involved; in particular, markers of oxidative stress and expression of calcium handling proteins. Based on differential observations in male and female offspring exposed to prenatal hypoxia regarding ex vivo and in vivo cardiovascular susceptibility [14,15,25], we investigated cardiac outcomes in both male and female offspring exposed to prenatal hypoxia following MI.

Materials and methods

Animal ethics

All study procedures were approved by the University of Alberta Health Sciences Animal Policy and Welfare Committee (protocol number AUP00000242) and were in accordance with the guidelines of the Canadian Council on Animal Care. All experimental protocols conformed to the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals (eighth edition, revised 2011).

Animal models

Female Sprague–Dawley rats (body weight: 250–275 g), obtained from Charles River, Quebec, Canada, were housed in a temperature-controlled room with a 10:14-h light-dark cycle. The animals were provided with free access to standard chow (Lab Diet, Ref. 5001 (3.02 kcal/mg; protein: 23%, fat: 4.5%, fiber: 6%) and water. After acclimatization for a week, they were mated overnight and pregnancy was confirmed (day 0) by the presence of sperm in a vaginal smear obtained the next morning. On day 15 of pregnancy, pregnant dams were individually housed from gestational day (GD) 15–21 in a plexiglass chamber where the oxygen concentration was maintained at either normoxic (21%) or hypoxic (11%) levels by the continuous infusion of nitrogen gas. Hypoxic dams were switched to normoxic environment at the end of GD 21, and both normoxic and hypoxic dams were allowed to give birth in a normal oxygen environment (21%). Normoxic or hypoxic offspring were obtained from 21 dams. After birth (GD 22), body weight, crown-to-rump length, and abdominal girth were recorded. At birth, litters were culled to eight pups (four males and four females) to control the postnatal environment. At 3 weeks of age, offspring were weaned and double-housed. At the time of weaning, male and female offspring were randomly assigned to undergo either sham or MI surgery. Thus, the experimental groups consisted of normoxia-sham, normoxia-MI, prenatal hypoxia-sham, and prenatal hypoxia-MI. Offspring were randomized to experimental protocols such that for each of the study parameters, no two animals came from the same litter.

MI surgery

Sham or MI surgery was performed in normoxic or prenatal hypoxic male and female offspring at the age of 12 weeks. Rats offspring were anesthetized (sedated with 3.5–4% isoflurane and maintained at 1.5–2.5% isoflurane in 100% oxygen with a flow of 1 l/min), intubated, and mechanically ventilated (respiratory rate of 80 cycles/min and tidal volume of 1.2 ml/100 g body weight). A left thoracotomy was performed at the fourth intercostal space and the left anterior descending (LAD) coronary artery was ligated between the pulmonary cone and the left auricle using 6/0 prolene suture. A successful ligation was confirmed by observation of a pale patch on the left ventricle (LV) below the ligation. The intercostal space and skin were closed using 4/0 silk suture. A sham surgery was performed using the same procedure without ligating the LAD coronary artery. The body temperature was maintained at 37°C using a heating pad throughout the procedure. Within 24 h of MI surgery, the percent survival was not significantly different in normoxia and prenatal hypoxia exposed male offspring (normoxia compared with prenatal hypoxia; 68.42% compared with 45.83%, P=0.216, Fisher’s exact test). Likewise, in female normoxia and prenatal hypoxia exposed offspring, the percent survival was not significantly different (normoxia compared with prenatal hypoxia; 57.89% compared with 68.00%, P=0.54, Fisher’s exact test). In male and female, normoxia and prenatal hypoxia exposed offspring, the percentage of animals with infarction after MI surgery was not significantly different between the groups (male, normoxia compared with prenatal hypoxia: 52.63% compared with 33.33%, P=0.23; female, normoxia compared with prenatal hypoxia: 42.10% compared with 40.00%, P=1.00).

Echocardiography

One week following sham or MI surgery, LV morphology (wall measurements, LV sizes, and volumes), systolic and diastolic functions were assessed using echocardiography in male and female offspring. All echocardiographic assessments were performed by a single operator. The echocardiography was performed in supine or semi-left lateral decubitus position using a 13–23 MHz linear array transducer on a high-resolution in vivo microimaging system Vevo 2100 (VisualSonics®, Toronto, ON, Canada) [27]. Animals were anesthetized (sedated with 4% isoflurane and 1 l/min compressed air and maintained at 1.5% isoflurane and 1 l/min compressed air) and echocardiography was performed in 2D guided B-mode and M-mode. The heart was imaged in the 2D mode in the parasternal long and short axis view with a depth of 2 cm. From this view, an M-mode cursor was positioned perpendicular to the anterior and posterior wall of the LV at the level of the papillary muscles. Wall measurements (left ventricular anterior wall thickness in diastole and systole, left ventricular posterior wall thickness in diastole and systole), LV size (left ventricular internal diameter (LVID) in diastole and systole), and LV volumes (end systolic and diastolic volumes (left ventricular end systolic volume (LVESV) and left ventricular end diastolic volume (LVEDV)), were obtained. According to Simpson’s (Simp) method, ejection fraction (EF) and fractional shortening (FS) were calculated using Simp volumes, Simp areas, and Simp lengths obtained from B-mode electrocardiogram-gated kilohertz visualization (EKV) images (recorded at proximal, middle, and distal level) as follows:

 
formula
 
formula

Volume; d and Volume; s were calculated as:

 
formula
 
formula

where; Dist, distal; Mid, middle; Prox, proximal.

In the apical four chamber view, the peak mitral flow velocities of the early rapid filling (E) wave and the late filling atrial contraction (A) wave, isovolumic contraction time (IVCT), ejection time (ET), isovolumic relaxation time (IVRT), E/A ratio, and deceleration time of E wave were obtained. Tei index was calculated as IVCT + IVRT/ET. Tissue Doppler imaging obtained from the mitral annulus was used to get mitral annular velocities (E′ and A′) which is a validated technique to assess diastolic dysfunction.

Measurement of infarct size

Hearts were excised and arrested in diastole in KCl (1 M) solution. The right ventricle and LV, including the septal wall, were dissected and separated. LV tissue was transversely cut 5–6 mm from the apex for infarct size determination. Previous studies have shown that determination of infarct size in this region corresponds linearly to mean infarct size of the entire heart [28,29]. The LV tissue was subsequently embedded in paraffin, sectioned at 10-μm thickness, and stained with Masson’s trichrome. The percentage of infarct size was calculated using length-based method as: the length of the infarcted segment at epicardial and endocardial surface/epicardial and endocardial circumference of LV section [30,31].

Immunofluorescence

LV tissues were embedded at optimum cutting temperature and sliced (9-µm thick) using a cryostat. Sections were fixed in cold acetone for 10 min and incubated overnight at 4°C in a humid chamber with the collagen type I (COL1A1) antibody (1:1000; Novus Biologicals, Canada) or rabbit polyclonal collagen type III (COL3A1) antibody (1:2000; Abcam). Sections were incubated with secondary antibody Alexa Fluor 488 donkey anti-rabbit (1:200; Thermo Fisher Scientific, Canada) or Alexa Fluor 546 goat anti-rabbit (1:200; Thermo Fisher Scientific, Canada), respectively for 1 h at room temperature in a humid chamber. Three to four random, non-overlapping image fields were captured using a 20× objective lens (200× magnification), and the average results were reported. An Olympus microscope IX81 (Olympus Canada Inc., Canada) with CoolSNAP HQ2 CCD camera (Photometrics, U.S.A.) operated by cellSens Dimensions 1.9 software (Olympus Canada Inc., Toronto, ON, Canada) was used for visualization and image capture. Images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, U.S.A.).

Dihydroethidium staining

Cardiac superoxide levels were quantitated using dihydroethidium (DHE) fluorescence as previously described [32]. Sections (10-µm thick) of non-infarcted LV tissue embedded at optimum cutting temperature were prepared using a cryostat. Sections were washed three times with Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, Canada) and incubated in a humid chamber at 37°C with 200 µM DHE (Biotium Inc., U.S.A.) in HBSS for 30 min. Sections were washed three times with HBSS, coverslipped, and visualized under fluorescence microscope. Three random, non-overlapping image fields were captured using a 10× objective lens (100× magnification), and the average results were reported. Images were captured using a fluorescence microscope (IX81 Olympus, Japan) with CoolSNAP HQ2 CCD camera (Photometrics, U.S.A.) operated by cellSens Dimensions 1.9 software (Olympus Canada Inc., Canada). Images were analyzed by ImageJ software (National Institutes of Health, Bethesda, MD, U.S.A.).

Western blot

Non-infarcted LV tissue (50–100 mg) homogenates were prepared in lysis buffer (in mM: 20 Tris (pH 7.4), 5 EDTA, 10 sodium pyrophosphate tetrabasic, 100 sodium, 9 fluoride, and 1% NP-40) containing protease (Protease Inhibitor Cocktail (1× Halt TM 200 protease inhibitor, 201 Thermo Scientific, U.S.A.) and 1 mM PMSF, Fluka Biochemika) and phosphatase inhibitors (2 mM sodium orthovanadate, Sigma). Total protein concentration of the lysate was determined by the BCA assay (Pierce, Rockford, IL, U.S.A.).

Total protein (100 µg) was loaded and separated on 7.5% SDS/polyacrylamide gels for SERCA2a and 4–15% gradient gels (Mini-PROTEAN TGX Precast gels, Bio–Rad, Mississauga, ON, Canada) for RYR2 and transferred on to a nitrocellulose membrane (0.2 µm; Bio–Rad, Mississauga, ON, Canada). Membranes were incubated with 50% blocking reagent for 1 h at room temperature. After washing with PBS solution, the membrane was incubated overnight at 4°C with primary antibodies for SERCA2a (1:2500, Santa Cruz Biotechnology), RYR2 (1:1000, Abcam), or β-actin (1:1000; Santa Cruz Biotechnology). The membrane was incubated with secondary antibody conjugated with the fluorescent tag (1:10000), blots were visualized with LI-COR Odyssey Bioimager and quantitated by densitometry with Odyssey V3.0 software (LI-COR Biosciences).

Statistical analysis

Results are presented as mean ± S.E.M. Statistical differences amongst the groups were assessed using a two-way ANOVA (sources of variation; prenatal hypoxia and MI) followed by Bonferroni multiple comparison post hoc tests or Fisher’s exact test using Prism 6 software (GraphPad Software, U.S.A.). A value of P<0.05 was considered significant.

Results

Fetal phenotypes at birth

The effect of prenatal hypoxia on fetal biometrics has been characterized by our group [15,16,22]. In the current cohort, prenatal hypoxia caused asymmetric growth restriction as indicated by reduced birth weight (16.87% in male offspring, P<0.01 and 18.47% in female offspring, P<0.01), and abdominal girth (4.95% in male offspring, P<0.05 and 4.84% in female offspring, P<0.05) with a similar crown-to-rump length (P>0.05).

Systolic function

In both male and female offspring, echocardiographic data demonstrated that there was an overall effect of MI on reducing EF and FS; indicating impaired systolic function following MI that was independent of prenatal hypoxia (Figure 1).

Cardiac systolic function

Figure 1
Cardiac systolic function

In vivo cardiac systolic function assessment using echocardiographic B-mode EKV images. Summary EF data in male (A) and female (C) offspring. Summary FS data in male (B) and female (D) offspring. All groups were compared using a two-way ANOVA followed by a Bonferroni post hoc test (n=5–7 offspring from different litters). ***P<0.001, ****P<0.0001 for differences in the main effect (P-hypoxia or MI), #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001 compared with normoxia-sham or prenatal hypoxia-sham. Abbreviation: P-hypoxia, prenatal hypoxia; MI, myocardial infarction.

Figure 1
Cardiac systolic function

In vivo cardiac systolic function assessment using echocardiographic B-mode EKV images. Summary EF data in male (A) and female (C) offspring. Summary FS data in male (B) and female (D) offspring. All groups were compared using a two-way ANOVA followed by a Bonferroni post hoc test (n=5–7 offspring from different litters). ***P<0.001, ****P<0.0001 for differences in the main effect (P-hypoxia or MI), #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001 compared with normoxia-sham or prenatal hypoxia-sham. Abbreviation: P-hypoxia, prenatal hypoxia; MI, myocardial infarction.

Diastolic function

In male offspring, an overall effect of MI was observed on parameters of diastolic function such as increased IVRT (P<0.05) and increased Tei index (P<0.001) (Table 1). Post hoc analysis revealed that the increased Tei index occurred primarily within the prenatal hypoxia offspring (Table 1, P<0.01). In mitral annular E′ and A′ velocities, there was a similar pattern of change with a significant interaction and prenatal hypoxia effect in both measures whereby MI caused an increase in normoxia offspring but a decrease in prenatal hypoxia offspring (Figure 2A,B). Interestingly, MI surgery caused a significant increase in the E/E′ ratio, an index of ventricular filling pressure, only in the prenatal hypoxia group (Figure 2C, P<0.01); indicating an increased susceptibility of prenatal hypoxia exposed male offspring to diastolic dysfunction following 1 week of MI.

Cardiac diastolic function

Figure 2
Cardiac diastolic function

In vivo cardiac diastolic function assessment using echocardiographic M-mode images. Summary data of mitral annular E′ velocity ((A) males, (D) females), mitral annular A′ velocity ((B) males, (E) females) and E/E′ ratio ((C) males, (F) females). All groups were compared using a two-way ANOVA followed by a Bonferroni post hoc test (n=5–7 offspring from different litters). *P<0.05, **P<0.01 for differences in the main effect (P-hypoxia or MI), #P<0.05, ##P<0.01 compared with normoxia-sham or prenatal hypoxia-sham, ††P<0.01 compared with normoxia-MI. Abbreviation: P-hypoxia, prenatal hypoxia; MI, myocardial infarction.

Figure 2
Cardiac diastolic function

In vivo cardiac diastolic function assessment using echocardiographic M-mode images. Summary data of mitral annular E′ velocity ((A) males, (D) females), mitral annular A′ velocity ((B) males, (E) females) and E/E′ ratio ((C) males, (F) females). All groups were compared using a two-way ANOVA followed by a Bonferroni post hoc test (n=5–7 offspring from different litters). *P<0.05, **P<0.01 for differences in the main effect (P-hypoxia or MI), #P<0.05, ##P<0.01 compared with normoxia-sham or prenatal hypoxia-sham, ††P<0.01 compared with normoxia-MI. Abbreviation: P-hypoxia, prenatal hypoxia; MI, myocardial infarction.

Table 1
Diastolic function in normoxia and prenatal hypoxia exposed offspring at 1 week post-MI or sham surgery
MeasurementNormoxiaPrenatal hypoxiaP-hypoxiaMIInt
ShamMIShamMI
Male offspring        
Mitral E max velocity, mm/s 826.77 ± 39.70 933.56 ± 30.59 847.24 ± 56.63 991.93 ± 131.83    
Mitral A max velocity, mm/s 694.15 ± 32.06 740.38 ± 125.56 601.62 ± 67.59 926.10 ± 199.36    
Mitral E/A index 1.18 ± 0.03 1.36 ± 0.17 1.44 ± 0.10 1.29 ± 0.04    
Mitral deceleration time, ms 32.03 ± 2.14 28.20 ± 2.45 38 ± 5.38 30.49 ± 1.25    
IVRT, ms 23.27 ± 0.78 25.30 ± 1.36# 23.23 ± 1.19 26.41 ± 1.23   
Tei index 0.61 ± 0.02 0.74 ± 0.04 0.65 ± 0.04 0.86 ± 0.03##  ***  
Female offspring        
Mitral E max velocity, mm/s 859.43 ± 46.65 815.65 ± 68.64 791.56 ± 48.11 810.15 ± 57.16    
Mitral A max velocity, mm/s 762.86 ± 44.00 614.70 ± 56.48# 542.38 ± 38.22 638.60 ± 58.66##   
Mitral E/A index 1.16 ± 0.03 1.34 ± 0.11 1.44 ± 0.10 1.24 ± 0.05   
Mitral deceleration time, ms 27.30 ± 1.42 26.26 ± 3.25 35.5 ± 12.58 26.85 ± 2.12   
IVRT, ms 24.60 ± 0.71 23.84 ± 1.55 24.48 ± 0.65 25.11 ± 0.89    
Tei index 0.63 ± 0.02 0.60 ± 0.02 0.58 ± 0.03 0.68 ± 0.05    
MeasurementNormoxiaPrenatal hypoxiaP-hypoxiaMIInt
ShamMIShamMI
Male offspring        
Mitral E max velocity, mm/s 826.77 ± 39.70 933.56 ± 30.59 847.24 ± 56.63 991.93 ± 131.83    
Mitral A max velocity, mm/s 694.15 ± 32.06 740.38 ± 125.56 601.62 ± 67.59 926.10 ± 199.36    
Mitral E/A index 1.18 ± 0.03 1.36 ± 0.17 1.44 ± 0.10 1.29 ± 0.04    
Mitral deceleration time, ms 32.03 ± 2.14 28.20 ± 2.45 38 ± 5.38 30.49 ± 1.25    
IVRT, ms 23.27 ± 0.78 25.30 ± 1.36# 23.23 ± 1.19 26.41 ± 1.23   
Tei index 0.61 ± 0.02 0.74 ± 0.04 0.65 ± 0.04 0.86 ± 0.03##  ***  
Female offspring        
Mitral E max velocity, mm/s 859.43 ± 46.65 815.65 ± 68.64 791.56 ± 48.11 810.15 ± 57.16    
Mitral A max velocity, mm/s 762.86 ± 44.00 614.70 ± 56.48# 542.38 ± 38.22 638.60 ± 58.66##   
Mitral E/A index 1.16 ± 0.03 1.34 ± 0.11 1.44 ± 0.10 1.24 ± 0.05   
Mitral deceleration time, ms 27.30 ± 1.42 26.26 ± 3.25 35.5 ± 12.58 26.85 ± 2.12   
IVRT, ms 24.60 ± 0.71 23.84 ± 1.55 24.48 ± 0.65 25.11 ± 0.89    
Tei index 0.63 ± 0.02 0.60 ± 0.02 0.58 ± 0.03 0.68 ± 0.05    

Values are expressed as mean ± S.E.M. (n=5–7 offspring from different litters). All groups were compared using a two-way ANOVA followed by a Bonferroni post hoc test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 for differences in the main effect (prenatal hypoxia or MI), #P<0.05, ##P <0.01 compared with normoxia-sham or prenatal hypoxia-sham, P<0.05 compared with normoxia-sham. Abbreviations: Int, interaction; IVRT, isovolumic relaxation time; P-hypoxia, prenatal hypoxia; MI, myocardial infarction.

In female offspring, there was an interaction effect in mitral A wave velocity whereby it was decreased significantly in normoxia-MI and increased in prenatal hypoxia-MI groups compared with their respective sham controls (Table 1). However, while there was an overall interaction effect (P<0.05), the E/A ratio did not change significantly in either normoxia-MI or prenatal hypoxia-MI groups (Table 1). An overall effect of MI on reducing mitral deceleration time was observed that was independent of prenatal hypoxia (Table 1). Similarly, an overall effect of MI was observed on increasing mitral annular A′ velocity independent of prenatal hypoxia (Figure 2D,E). In contrast with male offspring, the E/E′ ratio was not altered by prenatal hypoxia or MI in female offspring, indicating an absence of increase in ventricular filling pressure, one of the phenotypes observed in diastolic dysfunction (Figure 2F).

Cardiac morphometry and infarct size

In male offspring, echocardiography data showed that there was an overall effect of MI on decreasing anterior wall thickness, and increasing LVID and left ventricular volume during diastole and/or systole (Table 2). However, an increase in LVID during diastole was observed primarily in the prenatal hypoxia group indicating greater LV dilatation 1 week post-MI in male offspring exposed to prenatal hypoxia.

Table 2
Echocardiographic assessments in normoxia and prenatal hypoxia exposed offspring at 1 week post-MI or sham surgery
MeasurementNormoxiaPrenatal hypoxiaP-hypoxiaMIInt
ShamMIShamMI
Male offspring        
Body weight, g 530.13 ± 16.27 509.38 ± 10.34 517 ± 12.17 547 ± 16.54    
Basal heart rate, beats/min 337 ± 8 369 ± 17 374 ± 34 356 ± 5    
Wall measurements        
LVAW-end diastole, mm 1.80 ± 0.07 1.45 ± 0.20 1.89 ± 0.12 1.46 ± 0.15  **  
LVAW-end systole, mm 3.27 ± 0.16 2.03 ± 0.22### 3.36 ± 0.15 1.88 ± 0.1###  ***  
LVPW-end diastole, mm 1.98 ± 0.12 2.03 ± 0.07 2.08 ± 0.10 1.95 ± 0.10    
LVPW-end systole, mm 3.15 ± 0.15 2.82 ± 0.18 3.22 ± 0.12 3.17 ± 0.32    
LV size and volumes        
LVID-end diastole, mm 9.07 ± 0.11 9.98 ± 0.63 8.11 ± 0.15 9.93 ± 0.81#  **  
LVID-end systole, mm 5.16 ± 0.17 7.70 ± 0.63## 4.29 ± 0.3 7.31 ± 0.75##  ****  
LVEDV, μl 466.30 ± 14.2 567.98 ± 82.98 353.21 ± 16 527.88 ± 95.6   
LVESV, μl 128.65 ± 8.9 315.20 ± 69.6# 84.55 ± 16.1 301.6 ± 71#  ****  
Female offspring        
Body weight, g 324.5 ± 7.5 323.8 ± 14.9 303.2 ± 6.9 306.3 ± 7.9   
Basal heart rate, beats/min 366 ± 17 379 ± 16 314 ± 17 367 ± 10    
Wall measurements        
LVAW-end diastole, mm 1.61 ± 0.10 1.29 ± 0.20 1.45 ± 0.05 1.17 ± 0.09    
LVAW-end systole, mm 2.89 ± 0.14 1.78 ± 0.26### 2.84 ± 0.10 1.65 ± 0.17###  ****  
LVPW-end diastole, mm 1.70 ± 0.04 1.70 ± 0.07 1.81 ± 0.08 1.84 ± 0.13    
LVPW-end systole, mm 2.79 ± 0.08 2.67 ± 0.26 2.85 ± 0.23 2.64 ± 0.19    
LV size and volumes        
LVID-end diastole, mm 7.26 ± 0.25 8.02 ± 0.31 7.41 ± 0.15 7.94 ± 0.36    
LVID-end systole, mm 3.75 ± 0.26 5.75 ± 0.33### 4.15 ± 0.24 6.20 ± 0.33###  ****  
LVEDV, μl 279.42 ± 20.59 342.33 ± 27.86 291.38 ± 12.7 331.94 ± 33.08    
LVESV, μl 63.43 ± 11.15 179.7 ± 20.5### 78.26 ± 10.35 203.5 ± 24.1### ****  
MeasurementNormoxiaPrenatal hypoxiaP-hypoxiaMIInt
ShamMIShamMI
Male offspring        
Body weight, g 530.13 ± 16.27 509.38 ± 10.34 517 ± 12.17 547 ± 16.54    
Basal heart rate, beats/min 337 ± 8 369 ± 17 374 ± 34 356 ± 5    
Wall measurements        
LVAW-end diastole, mm 1.80 ± 0.07 1.45 ± 0.20 1.89 ± 0.12 1.46 ± 0.15  **  
LVAW-end systole, mm 3.27 ± 0.16 2.03 ± 0.22### 3.36 ± 0.15 1.88 ± 0.1###  ***  
LVPW-end diastole, mm 1.98 ± 0.12 2.03 ± 0.07 2.08 ± 0.10 1.95 ± 0.10    
LVPW-end systole, mm 3.15 ± 0.15 2.82 ± 0.18 3.22 ± 0.12 3.17 ± 0.32    
LV size and volumes        
LVID-end diastole, mm 9.07 ± 0.11 9.98 ± 0.63 8.11 ± 0.15 9.93 ± 0.81#  **  
LVID-end systole, mm 5.16 ± 0.17 7.70 ± 0.63## 4.29 ± 0.3 7.31 ± 0.75##  ****  
LVEDV, μl 466.30 ± 14.2 567.98 ± 82.98 353.21 ± 16 527.88 ± 95.6   
LVESV, μl 128.65 ± 8.9 315.20 ± 69.6# 84.55 ± 16.1 301.6 ± 71#  ****  
Female offspring        
Body weight, g 324.5 ± 7.5 323.8 ± 14.9 303.2 ± 6.9 306.3 ± 7.9   
Basal heart rate, beats/min 366 ± 17 379 ± 16 314 ± 17 367 ± 10    
Wall measurements        
LVAW-end diastole, mm 1.61 ± 0.10 1.29 ± 0.20 1.45 ± 0.05 1.17 ± 0.09    
LVAW-end systole, mm 2.89 ± 0.14 1.78 ± 0.26### 2.84 ± 0.10 1.65 ± 0.17###  ****  
LVPW-end diastole, mm 1.70 ± 0.04 1.70 ± 0.07 1.81 ± 0.08 1.84 ± 0.13    
LVPW-end systole, mm 2.79 ± 0.08 2.67 ± 0.26 2.85 ± 0.23 2.64 ± 0.19    
LV size and volumes        
LVID-end diastole, mm 7.26 ± 0.25 8.02 ± 0.31 7.41 ± 0.15 7.94 ± 0.36    
LVID-end systole, mm 3.75 ± 0.26 5.75 ± 0.33### 4.15 ± 0.24 6.20 ± 0.33###  ****  
LVEDV, μl 279.42 ± 20.59 342.33 ± 27.86 291.38 ± 12.7 331.94 ± 33.08    
LVESV, μl 63.43 ± 11.15 179.7 ± 20.5### 78.26 ± 10.35 203.5 ± 24.1### ****  

Values are expressed as mean ± S.E.M. (n=5–7 offspring from different litters). All groups were compared using a two-way ANOVA followed by a Bonferroni post hoc test. *P<0.05, **P<0.01, ***P< 0.001, ****P<0.0001 for differences in the main effect (prenatal hypoxia or MI), #P<0.05, ##P<0.01, ###P<0.001 compared with normoxia-sham or prenatal hypoxia-sham. Abbreviations: P-hypoxia, prenatal hypoxia; MI, myocardial infarction; Int, interaction; LVAW, left ventricular anterior wall; LVEDV, left ventricular end diastolic volume; LVESV, left ventricular systolic volume; LVID, left ventricular internal diameter; LVPW, left ventricular posterior wall.

In female offspring, there was an overall effect of MI on decreasing anterior wall thickness, and increasing LVID and left ventricular volume only during systole (Table 2). In contrast with male offspring, MI did not lead to LV dilatation during diastole in normoxia or prenatal hypoxia exposed female offspring at 1 week post-MI (Table 2).

Ex vivo autopsy data revealed that there were no significant differences in relative heart weight and LV weight amongst the groups in either male or female offspring (Figure 3A,B,E,F). Histology data showed that male and female offspring from both normoxia and prenatal hypoxia exposed groups had a similar infarct size 1 week following MI (Figure 3 C,D,G,H). Following this observation, we assumed sex and prenatal hypoxia as sources of variation and performed a two-way ANOVA. This analysis showed that there was an overall effect of sex (P<0.01) on infarct size; female offspring having a smaller percent infarct size compared with their male counterparts. However, within the male and female groups, infarct size was similar between normoxia and prenatal hypoxia conditions (results not shown).

Ex vivo cardiac morphometry and infarct size

Figure 3
Ex vivo cardiac morphometry and infarct size

Heart weight and LV weight were recorded 1 week post-MI and expressed as heart weight per 100 g of BW in male (A,B) and female (E,F) offspring (n=5–8 offspring from different litters). Infarct size was quantitated using histological images of LV tissue sections stained with Masson’s trichrome and expressed as % of total LV length; representative images are shown ((C,D) male and (G,H) female, n=4–5 offspring from different litters). All groups were compared using a two-way ANOVA followed by a Bonferroni post hoc test. Abbreviations: BW, body weight; P-hypoxia, prenatal hypoxia; MI, myocardial infarction.

Figure 3
Ex vivo cardiac morphometry and infarct size

Heart weight and LV weight were recorded 1 week post-MI and expressed as heart weight per 100 g of BW in male (A,B) and female (E,F) offspring (n=5–8 offspring from different litters). Infarct size was quantitated using histological images of LV tissue sections stained with Masson’s trichrome and expressed as % of total LV length; representative images are shown ((C,D) male and (G,H) female, n=4–5 offspring from different litters). All groups were compared using a two-way ANOVA followed by a Bonferroni post hoc test. Abbreviations: BW, body weight; P-hypoxia, prenatal hypoxia; MI, myocardial infarction.

Collagen I and collagen III levels

Following the observation of diastolic cardiac dysfunction, we assessed collagen I and III levels in non-infarcted LV tissue. In both male and female offspring, immunofluorescence data demonstrated that there was no effect of prenatal hypoxia or MI on cardiac collagen I and collagen III levels (Figure 4). Similarly, the ratio of collagen I/III was not different amongst the groups in both sexes (results not shown).

Cardiac tissue collagen levels

Figure 4
Cardiac tissue collagen levels

Representative microscopic images of LV tissue sections immunostained using collagen I (COL1A1) antibody (green color) and collagen III (COL3A1) antibody (red color) in male (A,B) and female offspring (C,D). Data were collected from three independent experiments, and quantitated from images using fluorescence microscope were expressed as mean fluorescence. All groups were compared using a two-way ANOVA followed by a Bonferroni post hoc test (male and female, n=3 offspring from different litters). Abbreviation: P-hypoxia, prenatal hypoxia; MI, myocardial infarction.

Figure 4
Cardiac tissue collagen levels

Representative microscopic images of LV tissue sections immunostained using collagen I (COL1A1) antibody (green color) and collagen III (COL3A1) antibody (red color) in male (A,B) and female offspring (C,D). Data were collected from three independent experiments, and quantitated from images using fluorescence microscope were expressed as mean fluorescence. All groups were compared using a two-way ANOVA followed by a Bonferroni post hoc test (male and female, n=3 offspring from different litters). Abbreviation: P-hypoxia, prenatal hypoxia; MI, myocardial infarction.

Markers of cardiac oxidative stress

The levels of superoxide in LV tissue were measured to assess cardiac oxidative stress. Superoxide levels were significantly increased in only the prenatal hypoxia group (Figure 5A, P<0.01); indicating increased cardiac oxidative stress in male prenatal hypoxia offspring following MI. In contrast, cardiac superoxide levels were unaltered in female offspring (Figure 5B).

Cardiac superoxide levels

Figure 5
Cardiac superoxide levels

Representative microscopic images of LV tissue sections stained with DHE in male (A) and female (B) offspring. Data were expressed as mean intensity/cell after quantitation of images using a fluorescence microscope. All groups were compared using a two-way ANOVA followed by a Bonferroni post hoc test (male and female, n=3 offspring from different litters). *P<0.05 for differences in the main effect (P-hypoxia or MI), ##P<0.01 compared with prenatal hypoxia-sham, ††P<0.01 compared with prenatal hypoxia-MI. Abbreviation: P-hypoxia, prenatal hypoxia; MI, myocardial infarction.

Figure 5
Cardiac superoxide levels

Representative microscopic images of LV tissue sections stained with DHE in male (A) and female (B) offspring. Data were expressed as mean intensity/cell after quantitation of images using a fluorescence microscope. All groups were compared using a two-way ANOVA followed by a Bonferroni post hoc test (male and female, n=3 offspring from different litters). *P<0.05 for differences in the main effect (P-hypoxia or MI), ##P<0.01 compared with prenatal hypoxia-sham, ††P<0.01 compared with prenatal hypoxia-MI. Abbreviation: P-hypoxia, prenatal hypoxia; MI, myocardial infarction.

SERCA2a and RYR2 protein levels

In both male and female offspring, an overall effect of MI on reducing cardiac SERCA2a protein levels was observed (Figure 6A,C) with a more dramatic reduction being observed in the male offspring. Interestingly, in male offspring prenatal hypoxia increased cardiac RYR2 protein levels, which were then reduced by MI in both normoxia and hypoxia groups (Figure 6B). No significant effect of either prenatal hypoxia or MI was observed in female offspring (Figure 6D).

Representative Western blot images for cardiac SERCA2a and RYR2 protein levels

Figure 6
Representative Western blot images for cardiac SERCA2a and RYR2 protein levels

In male and female offspring, calcium handling proteins were measured in heart tissue lysates 1 week post-MI. Blots were quantitated using Odyssey software and expressed as SERCA2a/β-actin or RYR2/β-actin ratio ((A,B) male, (C,D) female). All groups were compared using a two-way ANOVA followed by a Bonferroni post hoc test (n=3 offspring from different litters). *P<0.05, **P<0.01, ***P<0.001 for differences in the main effect (P-hypoxia or MI), #P<0.05 compared with normoxia-sham or prenatal hypoxia-sham. Abbreviation: P-hypoxia, prenatal hypoxia; MI, myocardial infarction.

Figure 6
Representative Western blot images for cardiac SERCA2a and RYR2 protein levels

In male and female offspring, calcium handling proteins were measured in heart tissue lysates 1 week post-MI. Blots were quantitated using Odyssey software and expressed as SERCA2a/β-actin or RYR2/β-actin ratio ((A,B) male, (C,D) female). All groups were compared using a two-way ANOVA followed by a Bonferroni post hoc test (n=3 offspring from different litters). *P<0.05, **P<0.01, ***P<0.001 for differences in the main effect (P-hypoxia or MI), #P<0.05 compared with normoxia-sham or prenatal hypoxia-sham. Abbreviation: P-hypoxia, prenatal hypoxia; MI, myocardial infarction.

Discussion

Prenatal hypoxia, leading to IUGR, is one of the most common consequences of complicated pregnancies worldwide. The increased susceptibility of IUGR populations to CVDs in later life highlights the importance of an optimal in utero environment and its persistent effect on an individual’s later life’s cardiovascular health and disease [11,33]; drawing the attention of researchers and clinicians alike. As a result, the paradigm of risk factors for CVDs has shifted from the orthodox cardiovascular risks factors, being of postnatal origins, to the prenatal environment during pregnancy. In prenatal hypoxia exposed rat models, we have characterized adverse cardiovascular consequences of prenatal hypoxia in adult life using ex vivo experimentation where I/R injury, aging, or an HF diet were utilized as insults to assess cardiovascular health outcomes [13,1517]. Based on these findings, it is of clinical significance to identify the susceptibility of adult offspring exposed to prenatal hypoxia to develop in vivo cardiovascular dysfunction following a myocardial ischemic insult that closely mimics disease in the clinical setting. These investigations highlight the necessity of appropriate cardiovascular health action plans for specific targetting of susceptible IUGR populations.

In male and female rat offspring, there was a similar pattern of change in cardiac morphology during systole and cardiac systolic function following MI. However, we observed diastolic cardiac morphological changes only in male offspring where LV dilatation was exacerbated by prenatal hypoxia. Interestingly, a greater degree of diastolic dysfunction was observed following MI in male offspring exposed to prenatal hypoxia compared with their normoxia and female counterparts. These data indicate an increased susceptibility of prenatal hypoxia exposed adult male offspring to develop cardiac diastolic dysfunction following in vivo ischemic myocardial injury. In general, the pathological changes observed in males were greater than those in females and these data highlight the sex-dependent cardiac outcomes; implicating a necessity for sex-specific strategies directed toward improved clinical cardiovascular outcomes in susceptible IUGR populations.

CVDs are on the rise globally and are one of the principal reasons for increased morbidity, mortality, and cost to health care systems [34]. Ischemic heart disease is a major manifestation of CVD and consists of coronary heart disease that might lead to MI. The pathophysiological changes in MI include wall thinning, LV dilatation, and LV hypertrophy of non-infarcted regions [35]. Consistent with these findings, in male offspring we observed decreased anterior wall thickness and increased LV internal diameter during diastole in the MI group which was not affected by prenatal hypoxia. In contrast, female offspring were resistant to these wall morphological changes independent of prenatal hypoxia or MI; demonstrating improved cardiac structural outcomes following 1 week of MI. In fact, previous studies have shown that male mice also had adverse LV remodeling following MI, including increased LV dilatation, compared with females [36,37]. In the present study, the reason behind these differential observations in male and female offspring might be due to MI size as we observed that female offspring had a smaller percent infarct size compared with males. Previous studies have shown that structural alterations correlate positively with infarct size following acute MI [38]. Indeed, female offspring were shown to have a decreased infarct size following cardiac ischemic injury [39]. Furthermore, both experimental and clinical studies have shown that female offspring have a better tolerance to remodeling and the adaptive response to MI [37].

In both male and female offspring, we observed cardiac systolic dysfunction in both normoxia (male, EF ~44% and female, EF ~48%) and prenatal hypoxia exposed offspring (male, EF ~44% and female, EF ~52%) following 1 week of MI; indicating the absence of a heightened susceptibility of prenatal hypoxia exposed offspring to in vivo cardiac systolic dysfunction compared with normoxia counterparts. Prunier et al. [40] have shown mild diastolic dysfunction in nonfailing MI and severe diastolic dysfunction in congestive heart failure MI male rats following 1 month of MI. Interestingly, we observed a pronounced cardiac diastolic dysfunction in adult male offspring exposed to prenatal hypoxia 1 week post-MI; indicating an increased susceptibility of prenatal hypoxia exposed male offspring to diastolic dysfunction in the early stages of MI. In contrast, diastolic dysfunction was not as evident as in female offspring. The relative cardioprotection against MI in female offspring might be due to their reproductive age considering the clinical observation of a worse prognosis of MI in postmenopausal women [41]. Findings from several studies attribute the cardioprotection observed in female to endogenous female sex steroid hormones [4244]. A limitation in our study is that we did not control for the stage of the estrous cycle stage. It had been shown that estrous cycle stage did not affect infarct size or susceptibility to myocardial ischemic injury in female Sprague–Dawley rats [26]. Therefore, it is unlikely that the physiologic fluctuations in estrogen during ovulatory cycle confounded our experimental studies. However, further studies are warranted to investigate whether the observed cardioprotection in female offspring exposed to prenatal hypoxia is associated with endogenous female sex steroid hormones.

The pathology of diastolic dysfunction may involve alterations in cardiomyocyte function and extracellular matrix composition. One of the principal pathophysiological findings in diastolic dysfunction is myocardial stiffness; the mechanism for which is controversial. Some studies have suggested that there are significant changes in the extracellular matrix, especially in collagen tissues [45,46], whereas others have found changes in titin phosphorylation [4749]. In both male and female offspring exposed to prenatal hypoxia, we did not observe any alterations in collagen levels following 1 week of MI in non-infarcted LV tissue. Both temporal and regional differences in the expression of collagen levels after MI have been shown [50]; which might explain the absence of a change in collagen levels in the present study.

Alterations in cardiomyocyte function during diastolic dysfunction might be caused by an increase in cardiac oxidative stress [51] and intracellular Ca2+ homeostasis dysregulation [5254]. Abundant reactive oxygen species have been shown to be involved in the pathophysiology of CVDs including MI [18]. Increased reactive oxygen species generation during MI may play a critical role in cardiac hypertrophy, apoptosis, calcium homeostasis dysregulation, and correlate with adverse outcomes. Adult prenatal hypoxia exposed offspring, males in particular, with known cardiac oxidative stress [22], may be exposed to a higher risk of CVD following a myocardial ischemic insult. Furthermore, studies have shown that exposure to prenatal hypoxia leads to fetal cardiac and vascular oxidative stress and promotes adverse adult cardiovascular outcomes in adult male offspring such as vascular dysfunction, hypertension, and sympathovagal imbalance [23,55]. Interestingly, the adult cardiovascular pathophysiology programmed in utero is sex dependent as evident by several animal and human studies (reviewed in [12]). In fact, we observed increased superoxide levels in prenatal hypoxia exposed male offspring compared with their normoxic counterparts. Interestingly, superoxide levels were significantly increased in only male prenatal hypoxia exposed offspring at 1 week post-MI. This suggests that oxidative stress may be one of the multiple pathophysiological pathways responsible for the observed structural and functional cardiac phenotypes of diastolic dysfunction in male prenatal hypoxia-exposed offspring. However, we did not observe an increase in superoxide levels in the normoxia group at 1 week post-MI. It has been previously shown that there was a progressive increase in markers of oxidative stress 2 weeks post-MI [56]. Furthermore, increased markers of oxidative stress have been demonstrated in ovariectomized female rat hearts following 10 weeks of MI [57] and in patients with dilated and ischemic cardiomyopathy [58,59].

In adult male but not female IUGR rat offspring, Harvey et al. [20] found impaired Ca2+ handling due to alterations in sarcoendoplasmic reticulum properties and decreased cardiac RYR2 protein levels. We observed an overall effect of both MI and prenatal hypoxia on cardiac RYR2 levels in male but not female offspring. However, contrary to our expectation, only MI, and not prenatal hypoxia, decreased SERCA2a protein levels in both male and female offspring. These data demonstrate that cardiac SERCA2a and RYR2 levels were involved in the pathogenesis of MI in our animal models. However, the role of cardiac SERCA2a in the heightened cardiac diastolic dysfunction in male prenatal hypoxia exposed offspring was not evident. Further investigation into cardiac SERCA2a activity levels and other proteins involved in calcium homeostasis are warranted in order to delineate their roles in the susceptibility of prenatal hypoxia exposed male offspring.

In conclusion, the present study identified sex-specific cardiovascular outcomes following an acute myocardial ischemic insult in our animal model of prenatal hypoxia. Interestingly, male IUGR offspring demonstrated an increased susceptibility to develop cardiac diastolic dysfunction. The findings of the present study highlight the necessity of appropriate early life screening and strategies to identify preclinical phenotypes in order to intervene at the stage of preclinical diastolic dysfunction in this susceptible IUGR population.

Clinical perspectives

  • Cardiac susceptibility of prenatal hypoxia exposed adult male and female offspring to ischemic myocardial injury is not known.

  • We demonstrated that male but not female rat offspring exposed to prenatal hypoxia are more susceptible to develop heart failure involving diastolic dysfunction following acute MI in adult life compared with offspring from healthy pregnancies.

  • Early life screening and identification of strategies are necessary to pinpoint subclinical cardiac phenotypes in susceptible adult populations born following hypoxic pregnancies.

Funding

This work was supported by the Canadian Institutes of Health Research [grant numbers MOP 133566 (to S.T.D.), MOP 106617 (to J.R.D.B.)]; the Women and Children’s Health Research Institute through the generous contributions of the Stollery Children’s Hospital Foundation and the Royal Alexandra Hospital Foundation; the fellowship from Molly Towel Perinatal Research Foundation of Canada (to A.S.)]. J. Dyck is a Canada Research Chair in Molecular Medicine and S. Davidge is a Canada Research Chair in Maternal and Perinatal Cardiovascular Health.

Author contribution

A.S. contributed to the conception and design of the study, collection of data, analysis and interpretation of data, and drafted the manuscript. N.M. established the animal model, analyzed and interpreted the data. A.Q. contributed to collection and interpretation of data. J.S.M. contributed to the conception and design of the study and critical revision of the manuscript. S.T.D. and J.R.B.D. contributed to the conception and design of the study, interpretation of data, and critical revision of the manuscript.

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Abbreviations

     
  • CVD

    cardiovascular disease

  •  
  • EKV

    electrocardiogram-gated kilohertz visualization

  •  
  • DHE

    dihydroethidium

  •  
  • EF

    ejection fraction

  •  
  • ET

    ejection time

  •  
  • FS

    fractional shortening

  •  
  • GD

    gestational day

  •  
  • HBSS

    Hank’s balanced salt solution

  •  
  • HF

    high-fat

  •  
  • IUGR

    intrauterine growth restriction

  •  
  • IVCT

    isovolumic contraction time

  •  
  • IVRT

    isovolumic relaxation time

  •  
  • I/R

    ischemia/reperfusion

  •  
  • LAD

    left anterior descending

  •  
  • LV

    left ventricle

  •  
  • LVID

    left ventricular internal diameter

  •  
  • MI

    myocardial infarction

  •  
  • RYR2

    ryanodine receptor 2

  •  
  • SERCA2a

    sarcoendoplasmic reticulum Ca2+-ATPase2a

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