Osteopontin Promotes Left Ventricular Diastolic Dysfunction through a Mitochondrial Pathway

Abstract

Background:

Patients with chronic kidney disease (CKD) and coincident heart failure with preserved ejection fraction (HFpEF) may constitute a distinct HFpEF phenotype. Osteopontin (OPN) is a biomarker of HFpEF and predictive of disease outcome. We recently reported that OPN blockade reversed hypertension, mitochondrial dysfunction and kidney failure in Col4a3^−/− mice, a model of human Alport Syndrome.

Objectives:

Identify potential OPN targets in biopsies of HF patients, healthy controls and human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). Characterize the cardiac phenotype of Col4a3^−/− mice, relate this to HFpEF and investigate possible causative roles for OPN in driving the cardiomyopathy.

Methods:

Ogdhl mRNA and protein were quantified in myocardial samples from patients with HFpEF, HFrEF, and donor controls. OGDHL expression was quantified in hiPS-CMs treated ± anti-OPN antibody. Cardiac parameters were evaluated in Col4a3^−/− mice with and without global OPN knockout or AAV9-mediated delivery of 2-Oxoglutarate Dehydrogenase-Like (Ogdhl) to the heart.

Results:

Ogdhl mRNA and protein displayed abnormal abundances in cardiac biopsies of HFpEF (N = 17) compared with donor controls (N = 12; p <0.01) or HFrEF patients (N = 12; p <0.05). Blockade of OPN in hiPS-CMs conferred increased OGDHL expression. Col4a3^−/− mice demonstrated cardiomyopathy with similarities to HFpEF including diastolic dysfunction, cardiac hypertrophy and fibrosis, pulmonary edema, and impaired mitochondrial function. The cardiomyopathy was ameliorated by Opn^−/− coincident with improved renal function and increased expression of Ogdhl. Heart-specific overexpression of Ogdhl in Col4a3^−/− mice also improved cardiac function and cardiomyocyte energy state.

Conclusions:

Col4a3^−/− mice present a model of HFpEF secondary to CKD wherein OPN and OGDHL are intermediates, and possibly therapeutic targets.

Condensed Abstract:

Abnormal OGDHL expression was observed in cardiac biopsies of HFpEF patients compared to healthy donor or HFrEF groups. OGDHL levels were responsive to OPN activity in hiPS-CMs and rodent CMs. Cardiac structure and function analyses of Alport (Col4a3^−/−) mice that model monogenic kidney disease revealed, in addition to hypertension and renal dysfunction, a HFpEF-like phenotype that included pulmonary edema, diastolic dysfunction, cardiac hypertrophy, myocardial fibrosis and mitochondrial dysfunction. We identify pivotal roles for OPN and mitochondrial OGDHL in the pathophysiology and show that cardiac-specific overexpression of Ogdhl alleviated cardiac dysfunction and improved energetics, similar to global OPN knockdown.

Introduction

Heart failure with preserved ejection fraction (HFpEF) is a complex and increasingly prevalent syndrome accounting for more than 50% of all HF cases (1,2). Relative to HF with reduced ejection fraction (HFrEF), HFpEF is more prevelant in the elderly and more commonly associated with, and possibly driven by co-morbidities including systemic hypertension, obesity, diabetes mellitus, chronic kidney disease, and coronary artery and microvascular diseases (3–7). Because of the heterogeneous nature of HFpEF and its diverse underlying etiologies, pharmacological strategies including neurohumoral inhibition that are successfully used to treat HFrEF have not shown efficacy in large clinical trials of HFpEF (8–13). Rather it is proposed that more personalized treatment strategies are required that are tailored to individual HFpEF-specific signaling and phenotypic diversity as reflected by patient presentation and predisposition (1).

Epidemiological analyses suggest that patients with HFpEF in the presence of renal dysfunction represent a distinct phenotype (14,15). Consistent with this, phenomapping studies identified patients with chronic kidney disease (CKD), electrical and myocardial remodeling, pulmonary hypertension, and RV dysfunction as a subset of HFpEF patients that are at high clinical risk relative to other phenomapped groups (1). Animal models that accurately reproduce the clinical symptoms of different HFpEF subsets would be valuable to identify signaling intermediates and test for safety and efficacy of phenotype-specific interventions.

Recently we presented evidence that the pro-inflammatory cytokine Osteopontin (OPN) plays a causal role in the progression of CKD in Alport (Col4a3^−/−) mice, a model of autosomal Alport syndrome (16). Genetic disruption of the OPN gene in Alport mice ameliorated CKD and reversed systemic hypertension and mitochondrial dysfunction (16). Other work including our own described causal roles for OPN in cardiovascular disease and heart failure in humans and animal models where it has been labeled a “remodeling-specific marker” (17–21). Plasma levels of OPN are increased in HFpEF patients and predict outcome (22,23).

Here we establish that Col4a3^−/− mice recapitulate multiple features of HFpEF, phenotypes that are ameliorated by targeting OPN. Alport mice may represent a subset of HFpEF patients wherein CKD is a primary cause of HF.

Methods

Animals

Animal procedures were approved by the University of Miami IACUC. Col4a3^−/− mice on 129X1/SvJ background were from Jackson Lab and inter-bred with C57Bl/6 (Opn^−/−) and BALB/c mice at least 10 times as described previously (16). 129J mice were used to validate the HFpEF cardiac phenotypes. Disease progression in Col4a3^−/− mice is highly dependent on genetic strain (24). Therefore, we used the 129X1/SvJ strain for invasive hemodynamic studies, AAV9-Ogdhl gene therapy, and titin isoform expression. Equal gender numbers were used.

Experimental procedures and statistical analyses are described in detail in the Online Appendix.

Results

Col4a3^−/− mice develop diastolic dysfunction with preserved ejection fraction, impaired strain and pulmonary congestion

We recently reported that Col4a3^−/− mice develop systemic hypertension(16). To examine cardiac function in Col4a3^−/− mice, echocardiography and 2D-speckle tracking was implemented on 2-month old mixed genetic background animals and compared with wild type and Opn^−/− littermates (16). Echocardiography revealed LV diastolic dysfunction of Col4a3^−/− hearts. Isovolumetric relaxation time (IVRT) was prolonged from 16.4±1.27 ms in wild type to 25.69±1.71 ms in Col4a3^−/− mice (p<0.0001), indicating impaired LV relaxation in Col4a3^−/− mice (Figure 1A). Additionally, we found significantly increased early trans-mitral flow velocity to early mitral annulus velocity ratio (E/E’) from 28.47±1.4 to 40.1±2.84 (p<0.001) in the Col4a3^−/− mice (Figure 1B and Online Figure 1). Increased E/E’ indicates elevated LV filling pressure and pulmonary artery wedge pressure (PCWP). The E/A ratio (early to late ventricular filling velocities) was also significantly reduced corroborating impaired LV relaxation in Col4a3^−/− mice (p<0.01, Figure 1C). The myocardial performance index (MPI), also known as the Tei index was increased in Col4a3^−/− mice by 63% compared to the wild type mice (p<0.01) (Figure 1D). The Tei index is inversely related to function consistent with elevated Tei indices reported in HFpEF patients (25).

Figure 1: OPN deficiency ameliorates cardiac dysfunction in Col4a3^−/− mice.

A-D. Echocardiography of 2-month Col4a3^−/− mixed background mice shows diastolic dysfunction as indicated by a prolonged IVRT, increased E/E’, reduced E/A, and elevated MPI. E-F. GLS and GCS were impaired in the Col4a3^−/− mice indicating subclinical systolic dysfunction. G Col4a3^−/− mice show preserved EF. H. Pulmonary edema is suggested by increased body weight-normalized lung weight as well as by elevated Lung wet-to-dry (W/D) ratio, indicating failing hearts in Col4a3^−/− mice. Hetero-/homo-zygous deletion of OPN in Col4a3^−/− mice improves cardiac function and prevents pulmonary edema. Complete echocardiographic and morphometric measurements are presented in Online Tables 1 and 2. Data are mean±SEM. N=10–37 mice per group. *p<0.05, **p<0.01, ***p<0.001 using student’s t-test for two groups (panel I) comparisons or one-way ANOVA with Tukey’s post hoc test for multiple groups (all other panels).

Myocardial strain analyses revealed altered myocardial deformation in Col4a3^−/− mice via impaired global longitudinal strain (GLS; −19.70±1.31 % in wild type versus −15.24±1.88 % in Col4a3^−/− mice, p<0.05) and global circumferential strain (GCS; −23.52±1.03 % in wild type versus −18.23±1.24 % in Col4a3^−/− group, P<0.01) as shown in Figure 1E–F, reflecting subclinical systolic dysfunction. This finding is also consistent with the subclinical systolic dysfunction reported in HFpEF patients (26). In addition, maximum opposing wall delay was increased in Col4a3^−/− hearts, indicating asynchrony (Online Table 1). Col4a3^−/− mice did not show significantly decreased EF, Cardiac Output or Stroke Volume (Figure 1G and Online Table 1). Col4a3^−/− mice showed a significant increase in normalized lung weight, as well as lung wet-to-dry weight ratio consistent with pulmonary congestion, and suggesting congestive heart failure (Figure 1H–I; p<0.01). Complete echocardiographic and morphometric measurements are presented in Online Tables 1 and 2.

Col4a3^−/− mice develop cardiac hypertrophy and fibrosis

Cardiac hypertrophy is common in HFpEF patients; therefore, we quantified cardiac dimensions in Col4a3^−/− mice. Echocardiographic measurements revealed significantly increased relative wall thickness, LV anterior wall thickness, and interventricular septum thickness, (p<0.05, Online Table 1) indicating gross hypertrophy. Additionally, wheat germ agglutinin (WGA) staining confirmed increased myocyte cross-sectional area in Col4a3^−/− mice compared to controls (p<0.001, Figure 2A–B). We next evaluated fibrosis, a major contributor to HFpEF. Picrosirius Red staining demonstrated a 4.2-fold increase in collagen content of Col4a3^−/− hearts compared to wild type controls (p<0.001; Figure 2C–D). In addition, incorporation of EdU, by Myosin Light Chain 2 (MLC2)-negative interstitial cells was significantly upregulated (Fig 2E–F) suggesting fibroblast proliferation. Likewise, the number of activated fibroblasts, as indicated by increased area of rough endoplasmic reticulum and collagen deposition, was increased (Electron Microscopy/EM; Figure 2G). These data establish the presence of cardiac hypertrophy and fibrosis in Col4a3^−/− mice.

Figure 2: OPN deficiency ameliorates cardiac hypertrophy and fibrosis in Col4a3^−/− mice.

A-B. WGA staining of Col4a3^−/− hearts shows cardiac hypertrophy in Col4a3^−/− mice as increased myocyte cross-sectional area that is reduced with OPN knockout. Scale bar: 20μm. N=3 mice per group. C-D. Picrosirius Red staining of cardiac cross-sections indicated increased Collagen deposition in Col4a3^−/− heart that is significantly reduced by OPN deficiency. Scale bar: 100μm. N=5–15 mice per group. E-F. The number of Edu positive interstitial cells (MLC2 negative) is elevated in Col4a3^−/− hearts but normalized with OPN deficiency Scale bar: 20μm. N=3 per mice group. G. OPN deficiency markedly decreased myocardial fibrosis in Col4a3^−/− mice as shown by EM images of reduced collagen fibers and interstitial “activated” fibroblasts. C: Collagen fibers, RER: Rough Endoplasmic Reticulum. Data are mean±SEM. *p<0.05, **p<0.01, ***p<0.001 using one-way ANOVA with Tukey’s post hoc test.

OPN deficiency in Col4a3^−/− mice ameliorates cardiac dysfunction and prevents hypertrophy and fibrosis

OPN is elevated in the circulation of HFpEF patients and predicts outcome ^22,23. We recently reported that Col4a3^−/− mice have increased renal and plasma levels of OPN (16). OPN is not upregulated in the hearts of Col4a3^−/− mice. To investigate the effects of OPN downregulation, Col4a3^−/− mice with homozygous and heterozygous deletion of OPN were subjected to cardiac functional and histological analyses. We found significant improvement in cardiac function and remodeling in Col4a3^−/− mice with OPN deficiency. OPN deletion ameliorated diastolic function by restoring IVRT, E/E’, E/A, and Tei index (Figure 1A–D). Moreover, Col4a3^−/− mice on hetero- or homo-zygous OPN knockout backgrounds showed normal myocardial wall thickness (Online Table 1) and myocyte cross-sectional area (Figure 2A–B). OPN deficiency markedly decreased myocardial fibrosis in Col4a3^−/− mice as shown by Picrosiurius Red staining (Figure 2C–D and Online Figure 2), reduced Edu incorporation in interstitial cells (Figure 2E–F), and reduced collagen fibers and interstitial “activated” fibroblasts (Figure 2G).

Dysregulated Ogdhl in Col4a3^−/− hearts

Gene microarray analysis were implemented on total RNA isolated from hearts of 2-month wild type, Col4a3^−/− and Col4a3^−/−Opn^−/− mice (n = 3 per group). We identified 19 differentially expressed genes, of which 3 were up-regulated and 16 downregulated (Figure 3A; Online Table 3). Quantitative real-time PCR (qPCR) results confirmed that the expression of Hbb-b1, Alas2, Cnn1, Aqp7 and Ogdhl genes was significantly lower in Col4a3^−/− hearts. In addition to mRNA expression, OGDHL protein levels were decreased in total homogenate and mitochondrial fractions of Col4a3^−/− hearts (Figure 3C–D) but increased in Col4a3^−/−Opn^−/− double knockouts (Figure 3F). OGDH enzymatic activity was also significantly lower in extracts from Col4a3^−/− hearts (Figure 3E). These data suggest that OGDHL regulation by OPN contributes to the cardiac pathology of Col4a3^−/− mice.

Figure 3: OGDHL is dysregulated in Col4a3^−/− hearts and normalized by OPN deficiency.

A. Microarray heatmap shows genes that are altered in the Col4a3^−/− hearts and upregulated with OPN deficiency. B. The downregulation of selected genes in the Col4a3^−/− hearts is validated by qPCR. Target validation in whole heart tissue (C) or mitochondrial fraction (D) shows reduced OGDHL protein and suppressed OGDH activity (E). F. OGDHL protein levels are upregulated with OPN knockout in the Col4a3^−/− hearts. Data are mean±SEM. N=3–5 hearts per group. *p<0.05 and **p<0.01 using unpaired Student’s t-test.

Increased oxidative stress and loss of mitochondrial integrity in Col4a3^−/− hearts

To further explore abnormalities in energy transduction we investigated mitochondrial morphology and function in Col4a3^−/− hearts. Elevated oxidative stress including depressed redox state and elevated lipid peroxidation is common in HF. We found that oxidative stress was markedly elevated in Col4a3^−/− hearts, including 50% reduction in GSH:GSSG ratio (Online Figure 3A) and 35% increase in the levels of malondialdehyde (MDA, Online Figure 3B). We also found significant reductions in the levels of the mitochondrial electron transport chain Complexes I, II and IV (p<0.05, Online Figure 3C). EM revealed dysmorphic mitochondria that were swollen with disorganized cristae in Col4a3^−/− hearts (Online Figure 3D). Such features of oxidative stress and mitochondrial dysfunction are common in HF patients and animal models (27–29).

Negative regulation of Ogdhl and mitochondria by OPN

To further investigate functional interactions between OPN and OGDHL we developed a monoclonal antibody against human OPN (OPN mAb). OGDHL protein was quantified in hiPS-CMs after treatment for 24h with OPN mAb. Immunostaining and western blots showed that neutralization of OPN conferred significant increases in OGDHL (Figure 4A–C). In addition, we found that treating mouse neonatal cardiomyocytes (nCMs) with recombinant OPN protein for 48 hours significantly suppressed ATP-linked oxygen consumption (Figure 4D–E). These data are consistent with negative regulation of Ogdhl and mitochondrial respiration by OPN.

Figure 4: OPN negatively regulates OGDHL in hiPS-CMs and respiration in nCMs.

A-C. Treatment of hiPS-CMs with OPN mAb significantly increases OGDHL protein levels by immunostaining and western blotting. N= 3 wells per group. D-E. Mouse neonatal cardiomyocytes treated with 20 μg/ml mouse recombinant OPN for 48 hours show significantly reduced ATP-linked oxygen consumption rate. N=9–16 wells per group. Data are representative of 3 independent experiments. Data are mean±SEM. *p<0.05, **p<0.01, ***p<0.001 using unpaired Student’s t-test.

Validation of diastolic dysfunction in Col4a3^−/− on 129J background

In order to validate the HFpEF cardiac phenotype of mixed background mice in pure 129J background, the experiments were repeated on Col4a3^−/− 129J mice. In contrast to the gross and cellular hypertrophy seen in Col4a3^−/− on a mixed background, Col4a3^−/− 129J mice did not demonstrate such hypertrophy (Online Table 4). This finding is consistent with previous reports (30,31). However, both echocardiography and invasive hemodynamic measurements confirmed diastolic dysfunction in Col4a3^−/− 129J mice similar to the mixed background results. As shown in Figure 5A–B, Col4a3^−/− 129J mice had significantly prolonged IVRT and markedly increased Tei indices. However, the Col4a3^−/− 129J mice displayed a more compromised systolic function than the mixed background mice. While EFs were not reduced (Figure 5C), Col4a3^−/− 129J mice showed significantly decreased cardiac output and stroke volume (Figure 5D–E), p<0.05). Moreover, strain analysis in Col4a3^-/−129J mice also indicated myocardial deformation shown by impaired GLS and GCS (Figure 5 F–G), similar to the mixed strain line. Complete echocardiographic and morphometric measurements are presented in Online Tables 4 and 5.

Figure 5: Cardiac dysfunction and impaired mitochondrial function in Col4a3^−/− mice are rescued by heart-specific Ogdhl overexpression.

A-G Cardiac function of Col4a3^−/− 129J mice was evaluated by echocardiography and strain analysis four weeks after AAV9-cTnt-Ogdhl or AAV9-cTnt-Luciferase injections showing improved MPI (B), improved strain (F,G) and reduced weight loss (H). Complete echocardiographic and morphometric measurements are presented in Online Tables 4 and 5. I-J. Extracellular flux assay confirmed suppressed mitochondrial respiration in adult cardiomyocytes isolated from Col4a3^−/− hearts. Four weeks post AAV9-cTnt-Ogdhl injections, the adult myocytes show significantly improved oxygen consumption rate (OCR). Data are mean±SEM. N=15–29 mice per group. *p<0.05, **p<0.01, ***p<0.001 using one-way ANOVA with Tukey’s post hoc test.

Invasive LV catheterization revealed markedly increased end-diastolic pressure-volume relationship (EDPVR, Figure 6B p<0.01), prolonged time constant of LV relaxation (Tau, Figure 6C, p<0.01) and significantly increased dP/dt_min (Figure 6D, p<0.05), each supporting diastolic dysfunction. Moreover, LV end-diastolic pressure (LVEDP) was increased from 4.6±0.3 in wild type mice to 8.63±1.01 in the Col4a3^−/− 129J group (Figure 6E, p<0.01), corroborating elevated LV filling pressures. Complete invasive hemodynamics measurements are presented in Online Table 6.

Figure 6: Pressure Volume Analysis of Col4a3^−/− 129J mice.

Invasive hemodynamic measurements demonstrated that 2-month Col4a3^−/− 129J background mice develop diastolic dysfunction. Complete invasive hemodynamic measurements are presented in Online Table 6. Data are mean±SEM. N=13–18 mice per group. *p<0.05, **p<0.01, ***p<0.001 using unpaired Student’s t-test.

While increased upregulation of titin N2BA isoform was reported in some HFpEF patients as a compensatory response to increased myocardial stiffness (32), it was absent in others (33). We detected no significant changes of titin isoforms in the LV of Col4a3^−/− hearts (Online Figure 4).

AAV9-Ogdhl gene therapy in Col4a3^−/− mice ameliorates cardiac dysfunction

We performed Ogdhl gene delivery to Col4a3^−/− hearts using heart-specific AAV9-cTnT-Ogdhl and AAV9-cTnT-Luciferase as control. Col4a3^−/− 129J mice at 4-weeks received single tail vein injections of AAV vectors (1×10¹² vg/mouse) and gene delivery was confirmed by IVIS imaging, qPCR and western blot after an additional 4 weeks (Online Figure 5). Cardiac function was analyzed in parallel by echocardiography. Although Ogdhl overexpression did not change the prolonged IVRT (Figure 5A), the myocardial performance index (MPI) was significantly reduced relative to un-injected or Luciferase-injected controls (Figure 5B). Moreover, Ogdhl overexpression markedly improved GLS (Figure 5F) relative to un-injected or Luciferase-injected controls, suggesting that gene therapy improved cardiac systolic function. Col4a3^−/− mice that underwent gene therapy also demonstrated significantly higher body weights relative to un-injected controls suggesting a generally improved physiology (Figure 5H). Complete echocardiographic and morphometric measurements are presented in Online Tables 4 and 5.

Compromised mitochondrial respiration is rescued by Ogdhl gene therapy

To directly assess myocardial mitochondrial respiration in Col4a3^−/− hearts ± Ogdhl, Seahorse XF assays were performed on adult cardiac myocytes 4 weeks after AAV9 delivery. We found that over-expression of Ogdhl in Col4a3^−/− mice significantly improved mitochondrial function (Fig 5I–J). These results support our hypothesis that cardioprotection conferred by OPN deficiency in Col4a3^−/− hearts is mediated at least in part through Ogdhl elevation and enhanced energy metabolism.

OGDHL expression is dysregulated in cardiac biopsies of HF patients

To determine whether mitochondrial functions and Ogdhl were similarly compromised in the clinical setting, we quantified Ogdhl transcript and protein levels in cardiac biopsies from patients with HFpEF, HFrEF and donor controls. Demographics of the patients are summarized in Table 1. Unexpectedly, we found that transcript levels of Ogdhl were significantly higher in samples from both HFpEF and HFrEF compared with healthy controls whereas OGDHL protein was elevated only in the HFpEF group (Figure 7). While these results do not support suppression of the Ogdhl gene as a general mechanism underlying global bioenergetic dysfunction during HF, they do suggest that this pathway is dysregulated in both HF groups relative to controls. It should be noted that obesity and diabetes were underlying co-morbidities of the HFpEF group relative to controls or HFrEF, and these may influence bioenergetic signaling pathways including Ogdhl expression. Therefore, the HFpEF phenotype of this group may be distinct from that driven primarily by CKD in patients or Alport mice.

Table 1.

Patient demographics.

	ALL			RNA			Protein
	Control (18)	HFpEF (27)	HFrEF (19)	Control (12)	HFpEF (12)	HFrEF (12)	Control (12)	HFpEF (17)	HFrEF (12)
Age (years)	57	63	57	60	64	58	57	63	59
% Female	50	56	32	50	67	8	42	47	50
% Caucasian	83	26	74	75	25	67	92	24	83
% ACE-I or ARB	17	59	63	25	67	67	0	53	67
% BB	17	67	95	25	83	92	8	59	100
% Insulin	6	37	16	0	50	8	8	35	17
% Diuretic	0	96	100	0	100	100	0	94	100
% HTN	39	96	100	50	100	100	25	94	100
% DM	17	63	21	17	67	17	8	65	17
% CAD	6	11	11	8	17	17	0	12	0
% Afib	6	30	58	8	33	50	0	29	83
BMI (kg/m2)	26.4	39.9	26.0	26.4	36.5	26.2	26.8	41.6	25.8
RAP (mmHg)		11	7		12	7		11	7
PCWP (mmHg)		19	18		20	17		18	19
CI (L/m/m2)		2.46	2.20		2.43	2.40		2.43	2.05
LVEF (%)	60	65	17	60	65	17	60	65	16

Figure 7: OGDHL expression is dysregulated in cardiac biopsies of HFpEF patients.

Ogdhl mRNA (A) and protein (B) levels are upregulated in HFpEF patients as shown by qPCR and western blotting, respectively. n.c.u.: Normalized Delta C, n.d.u.: Normalized Densitometry Unit. Data are mean±SEM. N=12–18 per group. *p<0.05, and **p<0.01, using Kruskal-Wallis nonparametric test with Dunn’s multiple comparisons test for qPCR and one-way ANOVA with Holm-Sidak’s post hoc test for western blots.

Discussion

Cardiac phenotype of Col4a3^−/− mice

We show that Alport (Col4a3^−/−) mice at age 2-months reproduce multiple phenotypes of HFpEF that were significantly ameliorated by genetic disruption of the OPN gene. Alport mice displayed diastolic dysfunction with preserved EF, myocardial deformation, hypertrophy, fibrosis, pulmonary edema, and mitochondrial dysfunction. Others and ourselves have previously reported on the renal insufficiency and systemic hypertension in these mice both of which are also significantly ameliorated by OPN deficiency (16,30). Despite their preserved EFs, Col4a3^−/− mice displayed myocardial deformation as indicated by impaired GLS and GCS that is suggestive of the subclinical systolic dysfunction often seen in human HFpEF (26,34).

Role of Osteopontin in driving a HFpEF-like phenotype

Col4a3^−/− animals with OPN hetero- or homozygous deletion presented a much more neutral cardiac phenotype with improved diastolic function and decreased myocardial hypertrophy and fibrosis. Microarray analysis identified multiple down-regulated, energetics-related genes in Col4a3^−/− hearts that were rescued by OPN deficiency. Of these, 2-Oxo-Glutarate Dehydrogenase-like (OGDHL), a mitochondrial protein involved in metabolic substrate fluxes and signaling (35,36), displayed the most robust response. OGDHL mRNA, protein and enzyme activity were all substantially decreased in Col4a3^−/− hearts, and OGHDL expression was rescued by OPN deficiency. Col4a3^−/− mice displayed dysmorphic and dysfunctional cardiac mitochondria with evidence of increased oxidative stress. These defects were also absent in OPN deficient Col4a3^−/− mice. Roles for OPN in disruption of mitochondrial functions in the model was further supported by our in vitro studies on isolated cardiac myocytes from mice or hiPS-CMs as well as by Ogdhl gene therapy in vivo. OPN reduced mitochondrial function of cultured cardiac myocytes and cardiac-specific overexpression of Ogdhl in Col4a3^−/− hearts reversed the mitochondrial defects. Notably, Ogdhl gene therapy conferred significant improvements of cardiac systolic function, strain, and bioenergetics, consistent with a central role for OPN-induced mitochondrial dysfunction in promoting a HFpEF-like cardiac phenotype in this model.

OGDHL protein levels were increased in cardiac biopsies of HFpEF patients. Depressed OGDHL activity has been associated with neuronal degradation (37) and tumor growth (38), whereas up-regulation of the enzyme has been described in association with stress in the brain and heart (39). Therefore, dysregulation of OGDHL in either direction associated with pathological stress may contribute to CKD-related HFpEF. Further studies are required to fully define the relationships between OPN, OGDHL and HF and determine whether the OGDHL/OPN axis is a viable therapeutic target in different subsets of HFpEF patients. Whereas our cellular and gene therapy data showing induced expression of OGDHL by treatment of CMs with an OPN neutralizing antibody and partial reversal of cardiomyopathy by AAV9-Ogdhl (Figure 5) supports direct effects of OPN on OGDHL, we cannot rule out the possibility that global KO of OPN in this model confers a time-restricted cardioprotection by an as yet unidentified pathway(s).”

Do Col4a3^−/− mice model a CKD subtype of HFpEF?

Impaired renal function is a risk factor for developing HFpEF (15,40). Therefore, we considered whether the Alport mouse models a CKD-HFpEF subset. Epidemiology studies indicate that a CKD-HFpEF subset of patients present with more LV hypertrophy, a larger LV systolic functional deficit, impaired left atrial mechanics and RV dysfunction (15). Our echocardiography and 2D-speckle tracking studies confirmed impaired LV relaxation, elevated LV filling and pulmonary artery wedge pressures, increased myocardial performance index and normal EF. Strain analyses confirmed myocardial deformation with impaired global longitudinal and circumferential strain reflecting mild systolic dysfunction. Col4a3^−/− mice displayed global myocardial hypertrophy, fibrosis and pulmonary congestion consistent CHF. These changes are consistent with such a CKD-induced HFpEF phenotype. Shah et al recently proposed a personalized medical approach to the treatment of HFpEF wherein each specific phenotype is targeted by polypharmacology (1). Such an approach may benefit from phenotype-specific animal models that could be used to test for safety and efficacy of such drug combinations. For example, Col4a3^−/− mice could be used to test for safety and efficacy of combinations of diuretics, ACEI, ARBs, β-blockers, dobutamine, neprilysin inhibitors, and OPN antagonists (monoclonal antibody or aptamer).

In conclusion, Col4a3^−/− mice reproduce a CKD-HFpEF-like phenotype that includes renal and diastolic dysfunction, myocardial deformation, hypertension, cardiac hypertrophy, pulmonary congestion, fibrosis and bioenergetic deficit. The phenotype is globally ameliorated by downregulation of OPN and the cardiac phenotype appears to be driven at least in part by OPN-mediated loss of mitochondrial enzymes including Ogdhl.

Limitations of our study include imperfect matching of the subset of the clinical HFpEF population (backgrounds of diabetes, obesity, and no data on serum OPN) with the preclinical model of CKD-related HFpEF. Similarly, while the human tissues were obtained from a similar region of the RV, the patients do have altered co-morbidity with greater obesity in the HFpEF subjects for example. Heart transplantation is generally restricted to a BMI <35, whereas many of our HFpEF patients have higher values. Donor hearts will not have this level of obesity or diabetes as seen in HFpEF. While some HFrEF patients had DM, it was not as common as in HFpEF. Donor hearts lacked these features by definition. In addition, left ventricle biopsies were taken from mice, although the anatomical mismatching may not be a major concern because in separate analyses, we did examine RV versus LV OGDHL protein expression in HFrEF and non-failing donors where we could obtain tissue from both ventricles from the same heart, and found them similar (Online Figure 6; Online Table 7).

Another limitation involves the short lifespan and severe nephropathy of the preclinical model. Finally, whereas bioenergetic perturbations involving mitochondrial Ogdhl are implicated by our HFpEF-Alport model studies, the discrepancy of OGDHL changes in human versus mouse samples leaves open the possibility that OPN confers its effects by as yet unidentified pathways possibly involving more global effects of the cytokine on heart and kidney function.

Central Illustration: Osteopontin and Mitochondrial OGDHL in Diastolic Dysfunction.

The phenotype of a subset of patients with HFpEF including a possible central and causative role for Osteopontin (OPN) is mimicked in Alport (Col4a3^−/−) mice. Our studies show that OPN regulates mitochondrial 2-Oxoglutarate Dyhydrogenase (OGDHL) and myocardial bioenergetics in a preclinical model, and this at least partially drives the HFpEF phenotype. The results were validated in human induced pluripotent-derived cardiomyocytes (hiPS-CMs), and cardiac biopsies of HFpEF patients.

CLINICAL PERSPECTIVES.

Competency in Medical Knowledge:

The Col4a3^−/− mouse exhibits several major pathological features of heart failure with preserved ejection fraction (HFpEF) associated with chronic kidney disease (CKD). In this model, blockade of osteopontin reversed hypertension, mitochondrial dysfunction and kidney failure.

Translational Outlook:

Further investigation is needed to determine if targeting specific regulators of myocardial energetics could favorably impact the clinical manifestations of HFpEF.

Abbreviations:

EDPVR

End-Diastolic Pressure-Volume Relationship

HFpEF

Heart Failure with Preserved Ejection Fraction

HFrEF

Heart Failure with Reduced Ejection Fraction

IVRT

Isovolumetric Relaxation Time

LVEDP

Left Ventricular End-Diastolic Pressure

LVEF

Left Ventricular Ejection Fraction

OGDHL

2-Oxoglutarate Dehydrogenase-Like

OPN

Osteopontin

hiPSCs

Human-induced pluripotent Stem Cells

CMs

Cardiac Myocytes