Renovascular Hypertension Induces Myocardial Mitochondrial Damage, Contributing to Cardiac Injury and Dysfunction in Pigs With Metabolic Syndrome

Abstract

BACKGROUND

Renovascular hypertension (RVH) often manifest with metabolic syndrome (MetS) as well. Coexisting MetS and hypertension increases cardiovascular morbidity and mortality, but the mechanisms underlying cardiac injury remain unknown. We hypothesized that superimposition of MetS induces myocardial mitochondrial damage, leading to cardiac injury and dysfunction in swine RVH.

METHODS

Pigs were studied after 16 weeks of diet-induced MetS with or without RVH (unilateral renal artery stenosis), and Lean controls (n = 6 each). Systolic and diastolic cardiac function were assessed by multidetector CT, and cardiac mitochondrial morphology (electron microscopy) and myocardial function in tissue and isolated mitochondria.

RESULTS

Body weight was similarly higher in MetS groups vs. Lean. RVH groups achieved significant stenosis and developed hypertension. Mitochondrial matrix density and adenosine triphosphate production were lower and H₂O₂ production higher in RVH groups vs. Lean and MetS. Lean + RVH (but not MetS + RVH) activated mitophagy, which was associated with decreased myocardial expression of mitophagy-related microRNAs. MetS groups exhibited higher numbers of intermitochondrial junctions, which could have prevented membrane depolarization/activation of mitophagy in MetS + RVH. Cardiac fibrosis, hypertrophy (increased left ventricular muscle mass), and diastolic function (decreased E/A ratio) were greater in MetS + RVH vs. Lean + RVH.

CONCLUSIONS

MetS+RVH induces myocardial mitochondrial damage and dysfunction. MetS + RVH failed to activate mitophagy, resulting in greater cardiac remodeling, fibrosis, and diastolic dysfunction. Mitochondrial injury and impaired mitophagy may constitute important mechanisms and therapeutic targets to ameliorate cardiac damage and dysfunction in patients with coexisting MetS and RVH.

Graphical Abstract

Graphical Abstract.

Renovascular hypertension (RVH) is an important cause of secondary hypertension and renal failure commonly associated with several cardiovascular complications.¹ Subjects with RVH often manifest with metabolic syndrome (MetS),² a cluster of major cardiovascular risk factors, including obesity, hypertension, lipid abnormalities, and insulin resistance that affects over 30% of the general population.³ RVH and MetS induce myocardial damage,⁴ which fosters an increased risk for heart failure.¹ However, the mechanisms underlying cardiac injury in patients with coexisting RVH and MetS remain largely unknown.

Cardiomyocytes are highly dependent on adequate function of their mitochondria,⁵ which occupy approximately one-third of the cell volume.⁶ Cardiomyocyte mitochondria are arranged in packed strands that run between the myofibrils where they form aligned arrays of cristae, known as intermitochondrial junctions (IMJs), that provide the structural basis to enhance electrochemical coupling.⁷ These organelles produce 90% of cellular energy in the form of adenosine triphosphate (ATP), which is utilized to support the contraction–relaxation cycle within the myocardium.⁸ Mitochondria also regulate several important cellular functions, such as generation of reactive oxygen species (ROS), proliferation, survival, and apoptosis.⁹ Therefore, mitochondrial damage can directly contribute to cellular injury and death.

In recent years, mitochondrial abnormalities and dysfunction have been reported in several experimental models of hypertension and heart failure.¹⁰ We have previously shown that diet-induced MetS in swine induces myocardial mitochondrial damage and dysfunction, associated with altered cytoskeletal–mitochondria–sarcoplasmic reticulum interaction and subendothelial microvascular loss.^11,12 We have also shown that cardiomyocytes from pigs with RVH exhibit significant mitochondrial structural abnormalities and dysfunction, associated with myocardial tissue damage, oxidative stress, and fibrosis.^13,14 However, whether coexistence with MetS magnifies cardiomyocyte mitochondrial damage in RVH remains unknown. This study hypothesized that superimposition of MetS induces myocardial mitochondrial damage, aggravating cardiac injury and dysfunction in swine RVH.

METHODS

Juvenile female domestic pigs (n = 24) were studied for 16 weeks with the approval of The Institutional Animal Care and Use Committee. At baseline, animals were fed either a high-cholesterol/high-carbohydrate diet (MetS) containing (in % kcal) 17% protein, 20% complex carbohydrates, 20% fructose, and 43% fat and supplemented with 2% cholesterol and 0.7% sodium cholate by weight¹⁵ or standard pig chow (Lean) for the duration of the study.

Six weeks later, animals were anesthetized with 0.25 g of IM tiletamine hydrochloride/zolazepam hydrochloride (Telazol/Fort Dodge) and 0.5 g of xylazine. Anesthesia was maintained with intravenous ketamine (0.2 mg/kg/minute) and xylazine (0.03 mg/kg/minute). Renal artery stenosis was induced in 6 Lean and 6 MetS pigs (Lean + RVH and MetS + RVH, respectively) by placing an irritant coil in the renal artery, as previously described,¹⁶ whereas a sham procedure was performed in the remaining 6 Lean and 6 MetS pigs (Lean + Sham and MetS + Sham, respectively).

Ten weeks later, animals were similarly anesthetized, the degree of stenosis in each animal determined by angiography, and systemic blood samples were collected for lipid panels (enzyme immunoassay kit), serum creatinine, and fasting glucose and insulin levels. Insulin resistance was assessed by the homeostasis model assessment of insulin resistance (HOMA-IR). Cardiac remodeling, as well as systolic and diastolic function were assessed using multidetector computed tomography (MDCT). Blood pressure was measured with an intra-arterial catheter during MDCT studies.

Three days after completion of in vivo studies, pigs were euthanized with an i.v. bolus of 100 mg/kg of sodium pentobarbital (Vortech Pharmaceuticals). Hearts were harvested and left ventricular (LV) tissue sections frozen in liquid nitrogen (and maintained at −80 °C) or preserved in formalin or Trump’s fixative for ex vivo studies.

Cardiac function

Cardiac function was assessed using MDCT (Somatom Sensation-128, Siemens), as previously described.¹⁷ The entire LV was scanned 20 times throughout the cardiac cycle to measure ejection fraction, whereas LV endocardial and epicardial borders were traced to calculate LV muscle mass. Images analyzed with the Analyze software package (Mayo Clinic). Early (E) and late (A) ventricular filling rates were calculated from the positive slopes of volume/time curves and E/A ratio calculated using MATLAB (Math-Work).

Ex vivo studies

Mitochondrial content, morphology, and function

Myocardial mitochondrial morphology was assessed by transmission electron microscopy (Philips-CM10). LV samples (2–3 mm³) were fixed with Trump’s solution and processed at our electron microscopy core facility. Ten representative cardiomyocytes were randomly selected in each pig myocardium and only mitochondria fully contained within the borders of the transmission electron microscopy images were analyzed.¹¹ Mitochondrial density (number of mitochondria/field) and matrix density (1/mean gray values) were measured using the “freehand tool” of Image-J. In addition, IMJs, specialized mitochondria–mitochondria interactions that sustain mitochondrial membrane potential among adjacent mitochondria,^18,19 were analyzed and quantified using Image-J. IMJ number was calculated in 10 representative cardiomyocytes by counting the number of IMJ/field and results averaged. IMJ electron density (1/mean gray values in arbitrary units) was calculated by tracing regions of interest around 10 representative IMJs randomly selected in each cardiomyocyte, and cristae spatial density by counting the number of cristae/IMJ. IMJ cristae alignment was estimated by measuring the angles between apposed cristae, where 180 degree angles represent “perfect alignment” and 90 degree angles “orthogonal alignment.”  ¹⁸

Myocardial mitochondria were also isolated using the MITO-ISO kit (ScienCell). Mitochondrial protein concentration was determined using the Pierce BCA Assay kit (Thermo Fisher), and mitochondrial membrane depolarization analyzed using flow cytometry (FlowSight/Amnis). Briefly, 100 µg of isolated mitochondria were incubated with JC-1 dye (Thermo Fisher) at a concentration of 1.5 µg/mg protein in a final volume of 1 ml. JC-1 dye exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~529 nm) to red (~590 nm). Consequently, mitochondrial membrane depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. In addition, mitochondrial hydrogen peroxide (H₂O₂) production was measured by colorimetric quantitative methods (BIOXYTECH), whereas ATP production (ATP/ADP) and cytochrome-c oxidase (COX)-IV activity were assessed by colorimetric and fluorometric methods (Abcam).

Mitochondrial homeostasis

Myocardial mitochondrial biogenesis evaluated by expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α (Abcam) and the transcription factor A mitochondrial (TFAM) (Cell Signaling), fusion by the expression of mitofusin (MFN)-1 (abnova) and optic atrophy protein (OPA)-1 (Santa Cruz), and fission by the expression of phosphorylated dynamin-related protein (pDRP)-1 (Cell Signaling),¹¹ which were normalized by the expression of COX-IV, as commonly done in studies exploring mitochondrial homeostasis.²⁰ Western blots were analyzed with a western blot imager (LAS 4010, GE) and quantified using ImageQuant TL 8.1, a cooled CCD camera for chemiluminescent western blot imaging that facilitates molecular weight determination. Mitophagy was assessed by double immunofluorescence staining with Parkin (Santa Cruz) and PTEN-induced kinase (PINK)-1 (Sigma). Parkin–PINK-1 colocalization was quantified with the Mander’s coefficient using Image-J, and reported as M1, which represents the overlap between Parkin and PINK-1. In addition, myocardial expression of PINK-1 (Invitrogen), BCL2 interacting protein (BNIP)-3 (Bioss), p62 (Abcam), and microtubule-associated protein 1A/1B-light chain (LC3B) (Abcam) were measured by western blotting, and normalized by GAPDH.

Mitophagy-related microRNAS

miRWalk2.0 was used to identify microRNAs capable of targeting the genes that encode for PINK-1 (PINK1), BNIP-3 (BNIP3), and LC3B (MAP1LC3B). Quantitative-polymerase chain reaction using the ΔΔCt method was used to evaluate random myocardial expression levels of ssc-miR-328 (PINK1), ssc-miR-22-3p (BNIP3), and ssc-miR-29b (MAP1LC3B), as well as ssc-miR-370, ssc-miR-320, and ssc-miR-490-5p (PINK1 and MAP1LC3B). microRNA expression levels were normalized to GAPDH.

Cardiac injury

Myocardial oxidative stress was assessed by in situ production of superoxide anion detected by dihydroethidium (Sigma).¹¹ Tubulointerstitial fibrosis was assessed in LV sections stained with trichrome, semi-automatically quantified in 15–20 fields, and the results from all fields averaged.¹³

Statistical analysis

Statistical analysis was performed using JMP Pro (SAS). Normally distributed data were compared using analysis of variance (ANOVA) with Tukey’s post hoc test. Non-normally distributed data were compared using nonparametric (Wilcoxon and Kruskal–Wallis) tests with Steel–Dwass post hoc test. Two-way ANOVA was performed to analyze the effects of the interaction of MetS diet and RVH, followed by Tukey’s test as appropriate. A simple linear regression analysis was performed to correlate myocardial mitochondrial ROS production and oxidative stress. R² was calculated by the least-squares fit. A P value ≤0.05 was considered statistically significant.

RESULTS

Body weight was equally elevated in MetS + Sham and MetS + RVH groups compared with their respective controls (Table 1). All RVH pigs developed significant renal artery stenosis of a similar degree. Mean arterial pressure was elevated in MetS + Sham and further increased in both RVH groups. Cholesterol fractions were higher in MetS compared with Lean groups, as were fasting insulin and HOMA-IR score. However, glucose levels were similar among the groups. Serum creatinine was higher in Lean + RVH vs. Lean + Sham and MetS + Sham pigs and further increased in MetS + RVH. Heart rate and ejection fraction did not differ among the groups. Yet, LV muscle mass was higher in Lean + RVH compared with Lean + Sham and MetS + Sham, but further increased in MetS + RVH. E/A ratio that decreased in Lean + RVH vs. Lean + Sham and MetS + Sham, further decreased in MetS + RVH. However, none of the parameters listed in Table 1 were influenced by the synergistic interaction of MetS diet and RVH (all P > 0.05, 2-way ANOVA).

Table 1.

Baseline characteristics and cardiac function in study groups (n = 6 each)

	Lean + Sham	MetS + Sham	Lean + RVH	MetS + RVH	Two-way ANOVA
					MetS × RVH
Body weight (kg)	61.5 (59.0–64.1)	81.5 (75.8–84.3)*	58.0 (51.3–61.2)†	85.1 (78.8–86.3)*  ,‡	0.71
Degree of stenosis (%)	—	—	78.6 ± 14.7*  ,†	79.6 ± 10.5*  ,†	0.91
MAP (mm Hg)	94.6 ± 7.2.0	105.4 ± 8.1*	117.9 ± 7.9*  ,†	116.8 ± 14.6*  ,†	0.16
Total cholesterol (mg/dl)	81.3 (74.8–87.6)	432.9 (342.3–490.1)*	82.5 (76.0–90.5)†	413.0 (302.2–557.8)*  ,‡	0.91
LDL cholesterol (mg/dl)	31.6 ± 6.6	414.8 ± 107.3*	33.4 ± 6.5†	363.3 ± 99.0*  ,‡	0.10
Triglycerides (mg/dl)	8.0 ± 1.2	18.5 ± 5.1*	8.7 ± 3.6†	13.3 ± 5.2*  ,‡	0.10
Fasting glucose (mg/dl)	127.5 ± 13.6	121.4 ± 13.5	129.5 ± 36.1	108.5 ± 31.4	0.48
Fasting insulin (mU/ml)	0.4 (0.4–0.5)	0.7 (0.7–0.8)*	0.4 (0.3–0.5)†	0.8 (0.7–0.8)*  ,‡	0.95
HOMA-IR score	0.6 (0.6–0.7)	1.4 (1.8–1.9)*	0.6 (0.6–0.7)†	1.9 (1.7–1.9)*  ,‡	0.48
Serum creatinine (mg/dl)	1.2 ± 0.3	1.2 ± 0.3	1.6 ± 0.2*  ,†	1.9 ± 0.2*  ,†,‡	0.23
Heart rate (beats/minute)	82.8 ± 17.5	75.5 ± 14.1	73.6 ± 6.7	84.1 ± 14.6	0.20
Ejection fraction (%)	50.9 ± 5.8	53.6 ± 7.7	53.6 ± 7.7	52.6 ± 8.1	0.85
LVMM (g)	122.3 ± 18.5	135.6 ± 6.8	150.8 ± 23.7*  ,†	175.2 ± 9.2*  ,†,‡	0.41
E/A ratio	1.2 ± 0.3	1.3 ± 0.3	0.9 ± 0.1*  ,†	0.7 ± 0.1*  ,†,‡	0.23

Abbreviations: ANOVA, analysis of variance; E/A, early (E) and late (A) ventricular filling; HOMA-IR, homeostasis model assessment of insulin resistance; LDL, low-density lipoprotein; LVMM, left ventricular muscle mass; MAP, mean arterial pressure; MetS, metabolic syndrome; RVH, renovascular hypertension.

*P < 0.05 vs. Lean + Sham; ^†P < 0.05 vs. MetS + Sham; ^‡P < 0.05 vs. Lean + RVH.

RVH induces myocardial mitochondrial structural abnormalities and dysfunction

Although myocardial mitochondrial spatial density was similar among the groups, matrix density equally decreased in Lean + RVH and MetS + RVH vs. Lean + Sham and MetS + Sham (Figure 1). The number, electron density, and cristae density of IMJs were higher in MetS groups compared with their respective controls (Supplementary Figure S1A). However, IMJ cristae alignment increased in Lean + RVH and MetS + RVH compared with Sham groups (Supplementary Figure S1B). JC-1 red/green fluorescence intensity ratio in isolated mitochondria decreased only in Lean + RVH (Figure 2a), whereas production of H₂O₂ was elevated in MetS + Sham, and further increased in Lean + RVH and MetS + RVH (Figure 2b). Mitochondrial COX-IV activity and ATP generation were reduced in MetS + Sham, and further decreased in Lean + RVH and MetS + RVH. Mitochondrial structure, IMJs, membrane potential, production of H₂O₂, COX-IV activity, and ATP generation were not influenced by the interaction of MetS diet and RVH (all P < 0.05, 2-way ANOVA).

Figure 1.

RVH induces myocardial mitochondrial structural abnormalities. Representative transmission electron microscopy and quantification of myocardial mitochondrial density and matrix density in Lean + Sham, MetS + Sham, Lean + RVH, and MetS + RVH pigs (n = 6/group). *P < 0.05 vs. Lean + Sham; ^†P < 0.05 vs. MetS + Sham. Abbreviations: MetS, metabolic syndrome; RVH, renovascular hypertension.

Figure 2.

MetS and RVH induce myocardial mitochondrial dysfunction. (a) Flow cytometric patterns of isolated mitochondria stained with JC-1 and quantification of red/green fluorescence intensity ratio (n = 6/group). (b) Myocardial mitochondrial hydrogen peroxide (H₂O₂) production, cytochrome oxidase (COX)-IV activity, and ATP/ADP ratio in study groups (n = 6/group). *P < 0.05 vs. Lean + Sham; ^†P < 0.05 vs. MetS + Sham; ^‡P < 0.05 vs. Lean + RVH. Abbreviations: MetS, metabolic syndrome; RVH, renovascular hypertension.

Lean + RVH activates mitophagy

Myocardial expression of PGC-1α was similar among the groups, but expression of TFAM was higher in Lean + RVH compared with Lean, and tended to be higher compared with MetS and MetS + RVH groups (Supplementary Figures S2 and S5), suggesting increased mitochondrial biogenesis. However, expression of MFN-1, OPA-1, and pDRP-1 was similar among the groups, indicating preserved mitochondrial dynamics. Myocardial PINK-1 expression and its colocalization with Parkin decreased in MetS + Sham, increased in Lean + RVH, and decreased to MetS + Sham levels in MetS + RVH, as did expression of LC3B (Figure 3a,b and Supplementary Figure S4). Expression of BNIP-3 tended to be higher in Lean + RAS compared with all other groups, but did not reach statistical significance, whereas expression of p62 did not differ among the groups (Figure 3b and Supplementary Figure S4). Myocardial biogenesis, dynamics, or mitophagy were not influenced by the interaction of MetS diet and RVH (all P < 0.05, 2-way ANOVA).

Figure 3.

Lean + RVH activates mitophagy. (a) Representative myocardial double immunofluorescence staining for PTEN-induced kinase (PINK)-1 (green) and Parkin (red), and their colocalization (yellow). Although Parkin expression was similar among the groups, PINK-1 expression and its colocalization with Parkin (M1 overlap coefficient) decreased in MetS + Sham, increased in Lean + RVH, and decreased to MetS + Sham levels in MetS + RVH (n = 6/group). (b) Myocardial expression of PINK-1, BCL2/adenovirus E1B 19 kDa protein-interacting protein (BNIP)-3, microtubule-associated protein light chain-3B (LC3B), and P62 in study groups (n = 6/group). *P < 0.05 vs. Lean + Sham; ^†P < 0.05 vs. MetS + Sham; ^‡P < 0.05 vs. Lean + RVH. Abbreviations: MetS, metabolic syndrome; RVH, renovascular hypertension.

Lean + RVH induces post-transcriptional regulation of mitophagy-related genes

microRNA target analysis revealed that PINK1, BNIP3, and MAP1LC3B could be targeted by a total of 165, 62, and 290 microRNAs, respectively (Supplementary Figure S3A). PINK1 and MAP1LC3B could be targeted by 21 common microRNAs, BNIP3 and MAP1LC3B by 4 common microRNAs, and PINK1 and BNIP3 by 2 common genes. Yet, no detectable microRNA is capable of targeting these 3 genes simultaneously. Myocardial expression of the PINK1 targeting microRNA ssc-miR-328, the BNIP3 targeting microRNA ssc-miR-122-3p, and the MAP1LC3B targeting microRNA ssc-miR-29b were lower in Lean + RVH compared with all other groups (Supplementary Figure S3B). Similarly, expression of ssc-miR-370, ssc-miR-320, and ssc-miR-490-5p, which target PINK1 and MAP1LC3B, were also exclusively reduced in Lean + RVH. However, expression of these microRNAs was not influenced by the interaction of MetS diet and RVH (all P < 0.05, 2-way ANOVA).

MetS + RVH magnifies cardiac injury

Myocardial superoxide anion production and fibrosis that increased in MetS + Sham and Lean + RVH compared with Lean + Sham, further increased in MetS + RVH (Figure 4a), but were not influenced by the interaction of MetS diet and RVH (both P < 0.05, 2-way ANOVA). Myocardial superoxide production (dihydroethidium) correlated directly with mitochondrial H₂O₂ production (Figure 4b).

Figure 4.

MetS + RVH aggravates myocardial oxidative stress and fibrosis. (a) Representative myocardium dihydroethidium (DHE) and trichrome staining (×40) and their quantification (n = 6/group), showing increased myocardial oxidative stress and fibrosis in MetS + Sham and Lean + RVH compared with Lean + Sham, which further increased in MetS + RVH. (b) Myocardial superoxide production (DHE) correlated directly with mitochondrial H₂O₂ production. *P < 0.05 vs. Lean + Sham; ^†P < 0.05 vs. MetS + Sham; ^‡P < 0.05 vs. Lean + RVH. Abbreviations: MetS, metabolic syndrome; RVH, renovascular hypertension.

DISCUSSION

Accumulating evidence suggest that mitochondrial structural abnormalities and dysfunction are implicated in the pathogenesis of hypertensive heart failure.¹⁰ Our previous studies in swine have shown that prior to any measurable changes in cardiac function, MetS alone disrupts the cytoskeletal–mitochondrial–sarcoplasmic reticulum architecture, leading to myocardial oxidative stress and microvascular loss.^11,12 Similarly, RVH alone in swine induces mitochondrial structural abnormalities and dysfunction, contributing to oxidative stress, fibrosis, and cardiac dysfunction.^13,14

In the current study, we took advantage of a novel swine model of coexisting MetS and RVH²¹ to test whether superimposition of MetS induces myocardial mitochondrial damage, leading to cardiac injury and dysfunction in swine RVH. The anatomy, physiology, and pathophysiology of the pig heart are very similar to humans.²² Anatomical, physiological, and hemodynamic properties of hypertension and cardiac damage in pigs are closer to humans compared with small animals.²³ Additionally, pigs allow collection of abundant myocardial tissue to quantify RVH-induced cardiac functional and structural injury. We opted for using female pigs to test whether the deleterious effects on MetS and RVH on the heart outweigh this sex-specific protection.^24,25 Our observations demonstrate that coexisting MetS and RVH induces myocardial mitochondrial damage, disclosed by fragmented cristae and reduced mitochondrial matrix density, associated with mitochondrial oxidative stress and impaired bioenergetics, reflected in increased production of H₂O₂, and decreased COX-IV activity and ATP generation.

Although, myocardial expression of PGC-1α was similar among the groups, Lean + RVH increased expression of TFAM, a mitochondrial DNA binding protein that regulates the packaging, stability, and replication of the mitochondrial genome.²⁶ Mitochondrial fusion (MFN-1, OPA-1) and fission (pDRP-1) activities remained unaltered. However, MetS and RVH induced important changes in mitophagy, the selective clearance of damaged mitochondria through autophagy.²⁷ This recycling process contributes to the preservation of cardiac homeostasis and often occurs to defective mitochondria following damage or stress. Accumulation of potentially cytotoxic mitochondria has been linked to progression of heart failure and their efficient degradation may exert cardioprotective effects.²⁸

Dissipation of mitochondrial membrane potential represents the initial step of this pathway. Depolarization of mitochondria triggers PINK-1 accumulation on the outer mitochondrial membrane,²⁹ and the subsequent recruitment of the cytosolic Parkin, which in turn increases the expression of the mitophagy protein receptor BNIP-3. This protein interacts with the ubiquitin-like protein LC3B, favoring the formation of an autophagosome that engulfs damaged mitochondria for their further degradation in lysosomes. BNIP-3 deficient mice accumulate damaged mitochondria and develop cardiac dysfunction,³⁰ whereas disruption of the BNIP3–LC3B interaction significantly decreases mitophagy.³¹

In this study, we found that expression of LC3B, and PINK-1, as well as PINK-1/Parkin colocalization decreased in MetS + Sham and MetS + RVH groups compared with Lean + Sham, suggesting that MetS decreased mitophagy. However, expression of the adaptor protein p62 was similar among the groups, arguing against a major regulatory role of this protein in our model. Contrarily, LC3B expression, PINK-1 expression and its colocalization with Parkin were higher in Lean + RVH compared with all other groups, and BNIP-3 expression tended to be higher as well, suggesting activation of mitochondrial autophagy. Despite subtle changes in the expression of BNIP-3, MetS, MetS + RVH, and particularly Lean + RVH, all induced important changes in myocardial mitophagy. Hence, Lean + RVH and MetS + RVH mitochondria exhibited a similar degree of mitochondrial injury, but only the former activated this protective mechanism to recycle damaged mitochondria. These observations suggest that MetS may blunt the ability of RVH to activate mitophagy. Although activation of mitophay often results in mitochondrial degradation and decreased mitochondrial density, increased mitochondrial biogenesis, partly by upregulation of TFAM, could have sustained mitochondrial number in Lean + RVH pigs.

Importantly, MetS- and RVH-induced cardiomyocyte mitochondrial damage might have contributed to cardiac injury, remodeling, and dysfunction. Myocardial production of ROS increased in MeS + RVH, in agreement with previous observations in failing human hearts.³² Myocardial superoxide anion production that increased in MetS + Sham and Lean + RVH further increased in MetS + RVH, and correlated directly with mitochondrial H₂O₂ generation. Mitochondrial ROS plays a critical role in cardiac hypertrophy and failure in murine models of hypertensive cardiomyopathy³³ and guinea pig models of nonischemic heart failure.³⁴ In agreement, increased myocardial oxidative stress in MetS + Sham, Lean + RVH, and MetS + RVH pigs could have contributed to interstitial fibrosis, and consequent cardiac hypertrophy (increased LV muscle mass), and diastolic function (decreased E/A ratio), common findings in patients with MetS⁴ and RVH.¹

We have previously shown that superimposition of MetS magnified renal tubular mitochondrial damage and altered mitochondrial homeostasis, blunting ATP production in the stenotic kidney.³⁵ Given that blood pressure was similarly elevated in RVH with or without MetS, loss of estimated glomerular filtration rate (GFR) might be an important determinant of cardiac damage in our model. Indeed, treatment strategies aimed to preserve renal function can ameliorate cardiac remodeling and improve cardiac function,³⁶ underscoring important interactions between the kidney and the heart.

Notably, the extent of cardiac remodeling, fibrosis, and diastolic dysfunction was greater in MetS + RVH compared with Lean + RVH, possibly partly due to the fact that MetS + RVH failed to activate mitophagy. PINK-1 deficient mice develop LV dysfunction and cardiac hypertrophy as early as 2 months of age.³⁷ Likewise, suppression of BNIP-3 in mice leads to accumulation of abnormal mitochondria and cardiac dysfunction.³⁰ Taken together, these observations suggest that myocardial mitochondrial injury and defective mitophagy might be implicated in the pathogenesis of MetS- and RVH-induced cardiac damage. Although activation of mitophagy in Lean + RVH failed to restore membrane potential, it resulted in lesser oxidative stress, fibrosis, and cardiovascular remodeling. Therefore, our findings suggest that both MetS and RVH can independently compromise myocardial mitochondrial structure and function, associated with cardiac remodeling and dysfunction. However, neither mitochondrial nor cardiac damage in our model were influenced by the interaction of MetS diet and RVH, implying additive rather than synergistic effects.

To further explore the mechanisms modulating myocardial mitophagy in MetS and RVH pigs, we examined IMJs. Previous studies have shown that neighboring mitochondria may exhibit coordination of inner mitochondrial membrane cristae independent from mitochondrial fusion events.^18,19 IMJs have been described in several cell types, but are highly abundant in cardiomyocytes due to their high energetic demand.¹⁸ In these electron-dense structures, adjacent mitochondria form aligned arrays of cristae, providing the structural basis to enhance electrochemical coupling.⁷ Therefore, intermitochondrial transmission of the electrochemical gradient may equilibrate membrane potential across adjacent mitochondria, preventing membrane depolarization and subsequent activation of mitophagy. The physical alignment of cristae across apposed mitochondria is critical for electrochemical gradient transfer, which is more efficient when cristae are oriented in parallel than in a perpendicular fashion.³⁸ In this study, we found that IMJ density, electron density, and cristae density were all higher in MetS compared with Lean groups, possibly secondary to increased metabolic demands, whereas alignment remained unchanged. Contrarily, IMJ cristae alignment similarly increased in Lean + RVH and MetS + RVH compared with Sham groups, whereas IMJ density, electron density, and cristae density were lower in Lean + RVH vs. MetS + RVH. Furthermore, mitochondrial membrane potential that remained unaltered in MetS + Sham and MetS + RVH groups compared with Lean + Sham, decreased only in Lean + RVH pigs. Therefore, mitochondrial coordination of cristae at IMJs might have prevented membrane depolarization in MetS + Sham and MetS + RVH, resulting in reduced mitophagy.

In addition, we measured myocardial expression of several mitophagy-related microRNAs. These small noncoding RNAs prevent the production of a particular protein by binding to and destroying the mRNA encoding for that protein. Mitophagy is highly regulated by microRNAs which can target several genes involved in different stages of mitochondrial degradation. For example, miR-204 prevents cardiomyocyte autophagy induced by ischemia–reperfusion by targeting LC3-II.³⁹ Likewise, miR-222 upregulates p62 and inhibits autophagy in endothelial cells.⁴⁰ Interestingly, this study found that myocardial expression of the microRNAs targeting PINK1, BNIP3, and MAP1LC3B was reduced in Lean + RVH pigs. Although, expression of these microRNAs in most MetS + RVH pigs was similar compared with Lean + RVH, a single MetS + RVH outlier precluded the significance compared with Sham groups. Thus, our results suggest post-transcriptional regulation of mitophagy-related genes particularly in Lean + RVH pigs. Yet, we cannot rule out that other factors, such as epigenetic changes or post-translational modifications could have modulated myocardial mitophagy in MetS and RVH pigs. Since patients with MetS and RVH exhibit myocardial damage,⁴ associated with an increased risk for heart failure,¹ interventions to preserve mitophagy may alleviate the cardiac effects of MetS and RVH in patients suffering from these conditions.

Our study has some limitations including the use of relatively young pigs, as well as the short duration of the disease. Nevertheless, myocardial injury and cardiac dysfunction in our swine MetS and RVH models are similar to humans,^1,4 enhancing the clinical relevance of our observations. Although our model of RVH showed a modest increase in mean arterial pressure, RVH with or without MetS instigates cardiomyocyte mitochondrial damage, associated with cardiac remodeling, fibrosis, and diastolic dysfunction. This is in line with our previous observation that cardiomyocytes from pigs with RVH without MetS exhibit significant mitochondrial damage and dysfunction, associated with myocardial tissue damage, oxidative stress, and fibrosis.^13,14 Therefore, despite their age, our pigs could be considered a suitable tool to assess the impact of coexisting MetS and RVH on myocardial mitochondrial. Further studies are needed to identify the long term effects of MetS and RVH on the myocardium.

In summary, our study shows for the first time that the combination of MetS and RVH is associated with myocardial mitochondrial damage, characterized by morphological abnormalities, increased production of ROS, and impaired energy production. Although Lean + RVH and MetS + RVH exhibited significant mitochondrial damage, only the former activated mitophagy, possibly in part due to Lean + RVH-induced post-transcriptional regulation of mitophagy-related genes or mitochondrial coordination of cristae at MetS + RVH IMJs. Importantly, myocardial mitochondrial damage was associated with LV remodeling, diastolic dysfunction, and cardiac fibrosis. Therefore our novel observations underscore the contribution of mitochondrial injury to MetS- and RVH-induced cardiac remodeling and dysfunction. Mitoprotective strategies have demonstrated important cardioprotective effects in experimental models of MetS^11,12 and RVH.^13,14 The current study extends previous observations and suggests that mitochondria may constitute a potential therapeutic target to ameliorate cardiac structural damage and dysfunction in patients with coexisting MetS and RVH.

FUNDING

This work was supported by the National Institutes of Health (NIH) grants: DK106427, DK122137, DK104273, DK120292, DK102325, and HL092621, and by the Mayo Clinic College of Medicine and Science.

DISCLOSURE

The authors declared no conflict of interest.