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Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease logoLink to Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease
. 2022 Dec 6;11(23):e027164. doi: 10.1161/JAHA.122.027164

Diabetes and Excess Aldosterone Promote Heart Failure With Preserved Ejection Fraction

Bence Hegyi 1,✉, Juliana Mira Hernandez 1,2, Christopher Y Ko 1, Junyoung Hong 1, Erin Y Shen 1, Emily R Spencer 1, Daria Smoliarchuk 1, Manuel F Navedo 1, Donald M Bers 1, Julie Bossuyt 1,✉
PMCID: PMC9851441  PMID: 36416174

Abstract

Background

The pathobiology of heart failure with preserved ejection fraction (HFpEF) is still poorly understood, and effective therapies remain limited. Diabetes and mineralocorticoid excess are common and important pathophysiological factors that may synergistically promote HFpEF. The authors aimed to develop a novel animal model of HFpEF that recapitulates key aspects of the complex human phenotype with multiorgan impairments.

Methods and Results

The authors created a novel HFpEF model combining leptin receptor–deficient db/db mice with a 4‐week period of aldosterone infusion. The HFpEF phenotype was assessed using morphometry, echocardiography, Ca2+ handling, and electrophysiology. The sodium‐glucose cotransporter‐2 inhibitor empagliflozin was then tested for reversing the arrhythmogenic cardiomyocyte phenotype. Continuous aldosterone infusion for 4 weeks in db/db mice induced marked diastolic dysfunction with preserved ejection fraction, cardiac hypertrophy, high levels of B‐type natriuretic peptide, and significant extracardiac comorbidities (including severe obesity, diabetes with marked hyperglycemia, pulmonary edema, and vascular dysfunction). Aldosterone or db/db alone induced only a mild diastolic dysfunction without congestion. At the cellular level, cardiomyocyte hypertrophy, prolonged Ca2+ transient decay, and arrhythmogenic action potential remodeling (prolongation, increased short‐term variability, delayed afterdepolarizations), and enhanced late Na+ current were observed in aldosterone‐treated db/db mice. All of these arrhythmogenic changes were reversed by empagliflozin pretreatment of HFpEF cardiomyocytes.

Conclusions

The authors conclude that the db/db+aldosterone model may represent a distinct clinical subgroup of HFpEF that has marked hyperglycemia, obesity, and increased arrhythmia risk. This novel HFpEF model can be useful in future therapeutic testing and should provide unique opportunities to better understand disease pathobiology.

Keywords: arrhythmia, diabetes, HFpEF, mineralocorticoid, SGLT2 inhibitor

Subject Categories: Animal Models of Human Disease, Basic Science Research, Calcium Cycling/Excitation-Contraction Coupling, Translational Studies, Electrophysiology

Nonstandard Abbreviations and Acronyms

AP

action potential

db/db+Aldo

db/db mice infused with aldosterone

APD

action potential duration

CaT

Ca2+ transient

db/db

leptin receptor–deficient mice (Leprdb/db)

HFpEF

heart failure with preserved ejection fraction

I Na,Late

late Na+ current

L‐NAME

Nω‐nitro‐L‐arginine methyl ester

SGLT2

sodium‐glucose contransporter‐2

SR

sarcoplasmic reticulum

WT

wild‐type

Clinical Perspective.

What Is New?

  • Diabetes and mineralocorticoid excess are synergistic pathogenic factors in promoting heart failure with preserved ejection fraction (HFpEF) phenotype.

  • Diabetes and aldosterone induce diastolic Ca2+ handling impairments and action potential duration prolongation and enhance late Na+ current, which promote diastolic dysfunction and arrhythmias in HFpEF.

  • The sodium‐glucose contransporter‐2 inhibitor empagliflozin reverses action potential duration prolongation and late Na+ current enhancement, acting directly on HFpEF cardiomyocytes.

What Are the Clinical Implications?

  • Patients with HFpEF and diabetic hyperglycemia may exhibit more severe diastolic dysfunction and have increased arrhythmia risk.

  • Patients with HFpEF and diabetes may benefit from drugs targeting mineralocorticoid signaling.

  • The sodium‐glucose contransporter‐2 inhibitor empagliflozin may have an important beneficial effect on cardiac electrical activity in patients with HFpEF and diabetic cardiomyopathy.

Heart failure with preserved ejection fraction (HFpEF) is a critical and unresolved public health concern because of its increasing prevalence, high morbidity and mortality, and limited clinical treatment options. 1 In addition to the characteristic diastolic dysfunction and common extracardiac comorbidities (eg, hypertension, diabetes, obesity, exercise intolerance, lung and kidney diseases), patients with HFpEF have longer QTc 2 and increased incidence of nonsustained ventricular tachycardia on ambulatory ECGs. 2 , 3 The risk for cardiac arrhythmias and sudden cardiac death may also be increased in HFpEF, 4 , 5 especially in patients with insulin‐treated diabetes. 6 Importantly, almost all drugs that provide benefit in patients with heart failure with reduced ejection fraction (EF) have failed clinical trials in HFpEF, with exceptions being the mineralocorticoid receptor antagonist spironolactone 7 and the sodium‐glucose cotransporter‐2 (SGLT2) inhibitors empagliflozin 8 and dapagliflozin. 9 However, further randomized HFpEF clinical trials are needed, and the exact molecular mechanisms of cardioprotective effects of these drugs remain incompletely understood. 10 Therefore, there is a pressing unmet need for better understanding of the disease pathophysiology and identification of novel therapeutic targets.

Progress in understanding and treating HFpEF has been hampered by limitations in preclinical animal models for HFpEF that fail to represent the full spectrum of the complex, multiorgan human HFpEF phenotype. 11 Moreover, multiple models may be needed to capture the clinically heterogeneous human HFpEF patient population. 1 , 12 , 13 Recently, 2‐ or multi‐hit models emerged to more closely recapitulate the human HFpEF syndrome. 11 These models include high‐fat diet–fed mice treated with the constitutive nitric oxide synthase inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME), 14 ZSF1 diabetic plus spontaneously hypertensive rats treated with the vascular endothelial growth factor‐2 inhibitor SU5416, 15 western diet–fed and aortic‐banded pigs, 16 and western diet–fed pigs treated with excess mineralocorticoid (deoxycorticosterone acetate). 17 Topical reviews highlighted that in addition to these multi‐hit models, the leptin receptor‐deficient db/db and aldosterone infusion models can each recapitulate human HFpEF to a certain degree. 10 , 18 However, db/db 19 , 20 or aldosterone infusion 21 alone may not induce severe diastolic dysfunction or multiorgan HFpEF phenotype. Here, we introduce a novel murine HFpEF model in which db/db mice are chronically infused with aldosterone (db/db+Aldo mice), thus combining marked metabolic alteration with mineralocorticoid excess. We hypothesized that this combination may synergize, leading to a robust HFpEF phenotype with marked diastolic dysfunction, increased proarrhythmia risk, and significant systemic multiorgan impairments. This model would complement the existing HFpEF models in having a more diabetic phenotype, because a one‐size‐fits‐all therapeutic strategy is unlikely to work in HFpEF. 1 Patients with diabetic HFpEF represent a large clinical pheno‐subgroup 22 with distinct myocardial gene expression profile, 23 impaired cardiomyocyte Ca2+ homeostasis, 24 and a particularly poor prognosis but better response to spironolactone therapy 22 compared with patients with nondiabetic HFpEF.

Here we show that db/db and chronic aldosterone infusion separately induced only very mild diastolic dysfunction without pulmonary congestion in mice. However, when these 2 factors are combined, db/db+Aldo mice exhibit marked diastolic dysfunction with preserved EF, cardiac hypertrophy, high levels of B‐type natriuretic peptide (BNP), and significant extracardiac comorbidities (morbid obesity, diabetes with marked hyperglycemia, pulmonary edema, and vascular dysfunction) in line with current human HFpEF diagnostic criteria. 11 At the cellular level, cardiomyocyte hypertrophy, prolonged Ca2+ transient (CaT) decay, and arrhythmogenic action potentials (APs) were observed in db/db+Aldo mice. Empagliflozin reversed the late Na+ current (I Na,Late) enhancement and proarrhythmic AP changes in db/db+Aldo, directly acting on cardiomyocytes despite that cardiomyocytes lack SGLT2 expression, 25 providing additional insights into myocyte drug targets and therapeutic benefits of empagliflozin. In conclusion, the db/db+Aldo model can be an important translational murine model of a more diabetic phenotype to complement existing animal models for studying disease pathobiology and future therapeutic testing in HFpEF.

METHODS

All animal handling and laboratory procedures were in accordance with the approved protocols (#21572 and 22834) of the Institutional Animal Care and Use Committee at University of California, Davis, conforming to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8th edition, 2011).

The data underlying this article will be shared on reasonable request to the corresponding authors.

Detailed methods are available in Data S1.

Animal Procedures

Twenty‐four adult (10‐week‐old, both sexes) Leprdb/db (stock #000697) and 24 corresponding wild‐type (WT) mice on C57BL6/J background were obtained from Jackson Laboratory. The animals were kept at standard temperature, humidity, and lighting. Food (Teklad, 2018) and drinking water were provided ad libitum. Osmotic minipumps (Alzet, model 2004) that delivered a continuous infusion of either d‐aldosterone (0.3 μg/h) 21 or vehicle (saline with 5% ethanol) for 4 weeks were implanted subcutaneously in 12‐week‐old mice (Figure 1A). We used block randomization with a block size of 4 animals (for 1 genotype and treatment group in 1 sex), with 16 control (WT+vehicle) and 16 two‐hit (db/db+Aldo) mice included (allowing for detailed isolated myocyte studies), and for the one‐hit controls we used 8 WT+Aldo and 8 db/db+vehicle mice. For proper allocation concealment, animals were recruited blinded based on sequential ear tag numbers randomly assigned by the animal housing facility. Each treatment group included an equal number of male and female animals. Enzymatic isolation of left ventricular (LV) cardiomyocytes was performed as previously described. 26

Figure 1. Robust heart failure with preserved ejection fraction (HFpEF) phenotype in db/db mice with chronic aldosterone infusion.

Figure 1

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A, Study protocol of HFpEF‐inducing treatment and assessment of cardiac function and multiorgan impairments. B, Plasma aldosterone levels 4 weeks after osmotic minipump implantation in WT and leptin receptor‐deficient db/db mice. C, Marked obesity, hyperglycemia, and hepatomegaly in db/db mice. D, Cardiac hypertrophy, pulmonary edema, and high BNP plasma levels in db/db+Aldo. Mean±SEM is shown. ANOVA followed by Tukey multiple comparisons test. Animal numbers are shown in the figure. BNP indicates B‐type natriuretic peptide; db/db+Aldo, db/db mice with chronic aldosterone infusion; HFpEF, heart failure with preserved ejection fraction; HM/TL, heart mass to tibia length ratio; and WT, wild‐type.

Blood Glucose, Aldosterone, and BNP Measurements

Blood glucose levels were measured in fresh blood samples collected from the middle tail vein using OneTouch UltraMini blood glucose monitoring system and test strips (LifeScan). BNP and aldosterone levels were measured in blood plasma by ELISA using manufacturers' instructions (RayBiotech, EIAM‐BNP‐1 and Cayman Chemical, Aldosterone EIA, respectively). Three technical replicates were performed for each biological sample.

Arterial Diameter Measurements

Freshly isolated mesenteric artery segments were mounted in a 5‐mL myograph chamber to determine arterial diameter changes in response to changes in intraluminal pressure as previously described. 27

Echocardiography

Transthoracic echocardiography was performed in anesthetized (isoflurane, 1%–3%) animals. LV M‐mode and Doppler images were acquired using a Vevo 2100 echocardiography system (FUJIFILM VisualSonics) equipped with a 40‐MHz transducer. Body temperature was carefully monitored, and anesthesia was adjusted to achieve heart rates of 350 to 450 beats per minute to assess diastolic dysfunction and 450 to 600 beats per minute to assess systolic cardiac function in each animal.

Calcium Imaging

Intracellular CaTs were measured using confocal microscopy in freshly isolated ventricular cardiomyocytes loaded with Fluo‐4 AM (10 μmol/L, 30 minutes; Invitrogen) at room temperature. 26

Cellular Electrophysiology

APs were recorded in isolated ventricular cardiomyocytes using patch‐clamp with physiological solutions at 37 °C. 26 Arrhythmogenic diastolic activities were assessed during a 1‐minute period following cessation of tachypacing. In voltage‐clamp experiments, I Na,Late was measured using 500‐millisecond depolarizing voltage pulses to −40 mV from −120 mV holding potential in every 5 seconds.

Statistical Analysis

Data are presented as mean±SEM. Statistical significance of differences was determined using t test, Mann–Whitney test, and ANOVA followed by Tukey or Dunn multiple comparisons test, when applicable. GraphPad Prism 9 was used for data analysis. P<0.05 was considered statistically significant.

RESULTS

Diabetes and Aldosterone Excess Induce Multiorgan Impairments Characteristic of HFpEF

Figure 1A illustrates the db/db+Aldo mouse protocol and measurements made at the end of 4‐week continuous aldosterone (or vehicle) infusion. Plasma aldosterone levels were elevated 2.5‐fold following 4‐week aldosterone infusion (versus vehicle) in both WT and db/db mice (Figure 1B). Morphometric evaluations (Figure 1C) showed morbid obesity, marked hyperglycemia, and hepatomegaly in all db/db mice (independent of aldosterone infusion) as expected in this genotype. However, only db/db+Aldo mice exhibited significant cardiac hypertrophy (P=0.011 for interaction between db/db genotype and aldosterone treatment in 2‐way ANOVA), pulmonary edema (P=0.037), and elevated BNP levels (P=1.2×10−5) as shown in Figure 1D. No major morphometric alteration was observed in aldosterone‐treated WT mice (WT+Aldo) versus vehicle‐treated WT controls (WT+vehicle).

Diabetes and Aldosterone Excess Induce Vascular Remodeling in HFpEF

Extracardiac comorbidities, including vascular abnormalities (vascular stiffening and endothelial and microvascular dysfunction) and hypertension, are frequently reported in patients with HFpEF. We carefully measured the arterial diameters and the myogenic response over a range of intravascular pressures (10 to 100 mm Hg) in isolated mesenteric arteries in WT+vehicle and db/db+Aldo mice (Figure 2A). Significant vascular remodeling in db/db+Aldo mice was evident from markedly increased myogenic tone (Figure 2B), which could contribute to an increased mechanical afterload on the heart.

Figure 2. Impaired arterial function in db/db mice with chronic aldosterone infusion.

Figure 2

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A, Increased arterial myogenic tone in db/db+Aldo (n=7 arteries from 5 animals) vs WT mice with vehicle infusion (WT+vehicle, n=5 arteries from 5 animals). Mean±SEM is shown. Two‐way repeated measures ANOVA with Geisser–Greenhouse correction. db/db+Aldo indicates db/db mice with chronic aldosterone infusion; and WT, wild‐type.

db/db+Aldo Mice Exhibit Marked Diastolic Dysfunction and Preserved EF

Echocardiographic evaluation (Figure 3) showed preserved fractional shortening and EF in WT+Aldo mice, whereas contractility was slightly reduced in db/db+vehicle mice. Importantly, fractional shortening and EF were preserved in db/db+Aldo mice (Figure 3A and 3B). Moreover, db/db+Aldo hearts exhibited significant concentric hypertrophy quantified as LV remodeling index (a ratio between LV mass/LV end‐diastolic internal diameter; P=1.2×10−5 for interaction between db/db and Aldo), whereas db/db+vehicle hearts showed only a small tendency for LV hypertrophy, and LV remodeling index was unchanged in WT+Aldo (Figure 3B). Indices of diastolic dysfunction (mitral E/A and E/e′) progressively increased during chronic aldosterone infusion (Figure S1), and, by the end of 4‐week treatment, both measures (Figure 3C) were markedly increased in db/db+Aldo mice (E/e′, P=1.6×10−8 for interaction between db/db and Aldo). In contrast, db/db+vehicle and WT+Aldo mice only showed slight increases in E/A and E/e′ over the 4‐week study period versus WT+vehicle (Figure 3C; Figure S2). Left atrial (LA) enlargement is a marker of diastolic dysfunction, 28 frequently observed in patients with HFpEF, 29 and LA area was also significantly increased in db/db+Aldo mice (P=0.001 for interaction between db/db and Aldo) but only slightly increased in db/db+vehicle and WT+Aldo mice (Figure 3C). These data suggest a synergy between diabetes and aldosterone signaling in promoting diastolic dysfunction.

Figure 3. Marked diastolic dysfunction with preserved systolic function in db/ddb/db+Aldo.

Figure 3

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A, Representative LV M‐mode, flow, and tissue Doppler echocardiographic images 4 weeks after aldosterone or vehicle minipump implantation in WT and db/db mice. B, Preserved FS and EF, and significantly increased LVRI in db/db+Aldo. C, Severe diastolic dysfunction and LA enlargement in db/db+Aldo. Mean±SEM is shown. ANOVA followed by Tukey multiple comparisons test. Animal numbers are shown in the figure. db/db+Aldo indicates db/db mice with chronic aldosterone infusion; E/A, ratio between mitral E wave and A wave; E/e′, ratio between mitral E wave and e′ wave; EF, ejection fraction; FS, fractional shortening; LA, left atrial; LV, left ventricular; LVAW, left ventricular anterior wall; LVID, left ventricular internal diameter; LVIDd, end‐diastolic left ventricular internal diameter; LVM, left ventricular mass; LVPW, left ventricular posterior wall; LVRI, LV remodeling index; and WT, wild‐type.

db/db+Aldo Mice Exhibit Diastolic Impairments in Cardiomyocyte Calcium Handling

Impaired cardiomyocyte Ca2+ handling can promote contractile dysfunction and arrhythmias. 30 , 31 Intracellular CaTs in db/db+Aldo myocytes stimulated at 1 Hz exhibited unchanged peak [Ca2+] i , elevated diastolic [Ca2+], and slowed decline of CaTs indicative of slower sarcoplasmic reticulum (SR) Ca2+ reuptake (Figure 4A through 4C). The elevated diastolic [Ca2+]i caused by slower CaT decline results in a trend toward smaller CaT amplitude. However, the caffeine‐induced CaT amplitude was unchanged, indicating similar SR Ca2+ content in db/db+Aldo versus control myocytes (Figure 4C).

Figure 4. Prolonged Ca transient decay in db/db+Aldo.

Figure 4

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A, Representative intracellular CaTs in WT+vehicle and db/db+Aldo cardiomyocytes at 1‐Hz pacing and following a rapid caffeine pulse (10 mmol/L). B, Intracellular Ca2+ levels quantified as changes in Fluo‐4 fluorescence. Diastolic [Ca2+] i is the ratio of minimum F between beats at 1 Hz and the resting F 0. C, Prolonged CaT decay tau and unchanged SR Ca2+ content in db/db+Aldo. Mann–Whitney test. Mean±SEM is shown. n=18 cells from 6 animals in WT+vehicle and n=21 cells from 8 animals in db/db+Aldo. Each individual myocyte is shown as a data point. CaTs indicates Ca2+ transients; db/db+Aldo, db/db mice with chronic aldosterone infusion; SR, sarcoplasmic reticulum; and WT, wild‐type.

db/db+Aldo Murine Cardiomyocytes Have Proarrhythmogenic Electrophysiological Changes

Because arrhythmias are more frequent in diabetic patients with HFpEF, we tested for proarrhythmic remodeling in db/db+Aldo mice. AP duration (APD) was markedly prolonged in db/db+Aldo mice (Figure 5A). APD prolongation was prominent at the later phase of repolarization (75% and 90% of repolarization) (Figures 5A; Figure S3). Moreover, short‐term temporal variability of APD was also markedly increased in db/db+Aldo (Figure 5B), which may reflect increased spontaneous Ca2+ release. 26 , 32 To further assess arrhythmia susceptibility, we tested spontaneous diastolic activities following a tachypacing protocol (1 minute at 10 Hz pacing). Delayed afterdepolarizations and spontaneous APs were significantly increased in db/db+Aldo mice (Figure 5C). Importantly, all proarrhythmic AP changes were reversed by empagliflozin pretreatment (1 μmol/L, 4 hours) in db/db+Aldo mice, while empagliflozin had no effect in WT+vehicle control mice (Figure 5A through 5C).

Figure 5. Arrhythmogenic AP changes in db/db+Aldo murine cardiomyocytes are reversed by empagliflozin.

Figure 5

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A, Representative APs in WT+vehicle and db/db+Aldo cardiomyocytes at 1‐Hz pacing. C m is shown in the inset (Mann–Whitney test). Prolongation of APD90 in db/db+Aldo is reversed by EMPA (1 μmol/L, 4 hours). B, STV of APD90 was increased in db/db+Aldo, and this increase was reversed by EMPA. In AP measurements, 16 cells from 7 animals in WT+vehicle without EMPA treatment; 23 cells from 7 animals in WT+vehicle with EMPA treatment; 18 cells from 8 animals in db/db+Aldo without EMPA treatment; and 23 cells from 8 animals in db/db+Aldo with EMPA treatment. C, DADs and spontaneous APs were increased in db/db+Aldo following cessation of tachypacing (10 Hz) and reversed by EMPA. In DAD measurements, 11 cells from 7 animals in WT+vehicle without EMPA treatment; 16 cells from 7 animals in WT+vehicle with EMPA treatment; 10 cells from 8 animals in db/db+Aldo without EMPA treatment; and 19 cells from 8 animals in db/db+Aldo with EMPA treatment. Mean±SEM is shown. ANOVA followed by Tukey multiple comparisons test. Each individual myocyte is shown as a data point. AP indicates action potential; APD90, AP duration at 90% repolarization; C m , cell capacitance; DADs, delayed afterdepolarizations; db/db+Aldo, db/db mice with chronic aldosterone infusion; EMPA, empagliflozin pretreatment; STV, short‐term variability; and WT, wild‐type.

Late Na+ Current Is Enhanced in db/db+Aldo Murine Cardiomyocytes

APD prolongation, predominantly at phase 3 repolarization (Figure 5; Figure S3), suggested a potential role for I Na,Late enhancement in db/db+Aldo myocytes. I Na,Late density (current amplitude normalized to cell capacitance) was markedly increased in db/db+Aldo (Figure 6). Importantly, empagliflozin preincubation (1 μmol/L, 4 hours) reversed I Na,Late upregulation in db/db+Aldo mice and had no effects in controls (Figure 6), in line with the significant effect of empagliflozin selectively on APD in db/db+Aldo versus WT+vehicle (Figure 5).

Figure 6. Empagliflozin reverses late Na+ current enhancement in db/db+Aldo murine cardiomyocytes.

Figure 6

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A, RepresentativeI Na,Late traces in WT+vehicle and db/db+Aldo myocytes without or with preincubation with EMPA (1 μmol/L, 4 hours) and subsequent acute TTX (10 μmol/L, 3 minutes) applications. (Peak I Na was off‐scale.) B, EMPA pretreatment reversed I Na,Late upregulation in db/db+Aldo. Mean±SEM is shown. ANOVA followed by Dunn multiple comparisons test. 12 cells from 4 animals in WT+vehicle without EMPA treatment; 13 cells from 4 animals in WT+vehicle with EMPA treatment; 12 cells from 4 animals in db/db+Aldo without EMPA treatment; and 12 cells from 4 animals in db/db+Aldo with EMPA treatment. Each individual myocyte is shown as a data point. db/db+Aldo indicates db/db mice with chronic aldosterone infusion; EMPA, empagliflozin pretreatment; I Na,Late, late Na+ current; TTX, tetrodotoxin; and WT, wild‐type.

DISCUSSION

Modeling Diabetic HFpEF in Preclinical Research

Diabetes and mineralocorticoid excess are associated with worse outcome in patients with HFpEF. 6 , 22 , 33 In preclinical research, db/db and aldosterone infusion models have been used independently to study HFpEF disease mechanisms. 19 , 21 However, db/db mice or aldosterone‐treated WT mice only exhibit a mild diastolic dysfunction (Figure 3), in line with previous reports, and fail to recapitulate the complex metabolic and hemodynamic derangements in HFpEF. 10 , 11 , 18 The cardiac systolic function tends to decrease with age in db/db mice 34 ; however, the additional hemodynamic challenge caused by excess aldosterone led to preserved EF in db/db+Aldo mice (Figure 3). In line with this, db/db mice did not develop heart failure with reduced EF following pressure overload induced by transverse aortic constriction. 35 This was associated with restoration of protein kinase D1 function, 35 which importantly regulates cardiac hypertrophy and progression to heart failure with reduced EF. 36 Aldosterone infusion alone induces mild changes in cardiac function unless accompanied by additional stressors (eg, uninephrectomy+salt water 21 or myocardial infarction). 37 Here we showed that combined db/db+Aldo synergistically induces marked diastolic dysfunction, concentric cardiac hypertrophy, pulmonary congestion, and multiple common comorbidities of HFpEF, including diabetes, obesity, and increased vascular resistance (Figures 1, 2, 3), more closely recapitulating important aspects of human HFpEF. Thus, our new 2‐hit HFpEF model (db/db+Aldo) complements other recent preclinical models with a somewhat different disease pathophysiology (eg, versus a model of nitrosative stress in high‐fat diet+L‐NAME–treated mice 14 ). Indeed, individual HFpEF animal models (including this db/db+Aldo model) may best phenocopy a different subset of patients with HFpEF, 12 , 22 , 23 , 24 each of which may benefit most by different targeted therapeutic strategies. Parallel use of these translational HFpEF models can help to better understand disease pathomechanisms, find new molecular targets, test new drugs, and stratify subgroups of patients with HFpEF with prognostic and therapeutic implications. 1 , 13

Mechanisms of Increased Arrhythmia Susceptibility in Diabetic HFpEF

The db/db+Aldo model showed proarrhythmic changes in Ca2+ handling (Figure 4) and electrophysiology (Figures 5 and 6), in line with the increased arrhythmia susceptibility in diabetic patients with HFpEF. 6 , 22 The QTc is longer in patients with HFpEF, 2 and an increased incidence of nonsustained ventricular tachycardia was reported on their ambulatory ECGs, 2 , 3 correlating with APD prolongation and increased delayed afterdepolarizations in db/db+Aldo mice (Figure 5). Diabetic hyperglycemia has been shown to induce a complex signaling network of oxidative stress, intracellular glycosylation, and activation of protein kinases (Ca2+/calmodulin‐dependent protein kinase II, protein kinase C, protein kinase D1), which impairs the function of multiple sarcolemmal and sarcoplasmic ion channels to promote proarrhythmic APs. 26 , 38 , 39 , 40 Noncardiomyocyte mechanisms, including inflammation, fibrosis, and coronary artery disease may further enhance arrhythmias in diabetic HFpEF. 38 We also show elevated arterial myogenic tone at physiological arterial pressures (Figure 2), which could contribute to hypertension observed in many patients with HFpEF. The increased mechanical afterload caused by hypertension can further enhance arrhythmogenic Ca2+ handling and ion channel functional impairments in cardiac myocytes, dependent on nitric oxide signaling. 41 , 42

Potential Ionic Mechanisms and Antiarrhythmic Effects of Empagliflozin in HFpEF

Empagliflozin reduces mortality and hospitalization in patients with HFpEF with or without diabetes. 8 In line with this, SGLT2 inhibitors were shown to provide direct cardiovascular benefits beyond glycemic control. 25 , 43 Moreover, SGLT2 expression is lacking in cardiomyocytes, 44 suggesting an off‐target effect. The sodium‐hydrogen exchanger has been suggested as a potential empagliflozin target in cardiomyocytes 45 ; however, this mechanism remains controversial. 46 Another target for SGLT2 inhibitors in cardiomyocytes can be the late Na+ current. 47 Recently, we showed that empagliflozin reversed the enhancement of late Na+ current and APD prolongation in a different HFpEF model induced by high‐fat diet+L‐NAME treatment. 48 However, this empagliflozin effect on late Na+ current and APD in HFpEF required drug preincubation for 4 hours and suggested that the effect could be mediated by reduction of oxidative stress and suppression of Ca2+/calmodulin‐dependent protein kinase II. 48 , 49 Here we confirm this therapeutic benefit of empagliflozin in an additional HFpEF model, showing a complete reversal of I Na,Late enhancement and proarrhythmic AP changes in db/db+Aldo cardiomyocytes (Figures 5 and 6). Thus, empagliflozin might have beneficial electrophysiological effects by reversing the cellular Na+ and Ca2+‐handling impairments, which form a vicious cycle promoting contractile dysfunction and arrhythmias in the failing heart. 31 While the potential antiarrhythmic effects of SGLT2 inhibitors in patients with HFpEF are yet to be determined, dapagliflozin reduced the risk of ventricular arrhythmias and sudden cardiac death in patients with heart failure with reduced EF. 50 Dapagliflozin also attenuated diastolic dysfunction in a similar mouse model presented here, which used chronic angiotensin II infusion in db/db mice. 51

Study Limitations

Aging, an important characteristic of patients with HFpEF, has not been considered in this animal model. 11 In addition to arrhythmogenic ventricular remodeling, atrial fibrillation is a frequent comorbidity in aging patients with HFpEF, 52 , 53 which requires further investigation. Sex differences and the underlying molecular mechanisms were not studied here; however, cardiac remodeling is more prominent in diabetic women with HFpEF, 54 and female db/db mice, 20 and mineralocorticoid receptor inhibition may provide more benefit in women with HFpEF. 55

CONCLUSIONS

In the current study, we showed that diabetes and excess aldosterone synergistically promote diastolic dysfunction, concentric cardiac hypertrophy, elevated BNP levels, and significant extracardiac comorbidities (including severe obesity, diabetes with marked hyperglycemia, pulmonary edema, and vascular dysfunction), recapitulating important aspects of human HFpEF. At the level of cardiac myocytes, diabetes and excess aldosterone induced diastolic Ca2+‐handling impairments and APD prolongation and enhanced I Na,Late, which could promote diastolic dysfunction and arrhythmias in this murine model of HFpEF. Empagliflozin reversed I Na,Late enhancement and cellular proarrhythmia, directly acting on murine HFpEF myocytes. In conclusion, the db/db+Aldo model represents an important clinical subgroup of HFpEF that has marked hyperglycemia and obesity and increased arrhythmia risk. This novel HFpEF model can be useful to better understand disease pathobiology and therapeutic effects.

Sources of Funding

This work was supported by grants from the National Institutes of Health: P01‐HL141084 (Bers), R01‐HL142282 (Bers and Bossuyt), and R01‐HL149127 (Navedo); and the Minciencias – Fulbright Colombia (Mira Hernandez).

Disclosures

None.

Supporting information

Data S1

Figures S1–S3

Click here for additional data file. (582.6KB, pdf)

Acknowledgments

We thank Mukul Sharda, Adam Wilder, Anastasia Krajnovic, Vicky Diep, and Megan Ngim for their help in animal care, tissue collection, and laboratory tasks.

For Sources of Funding and Disclosures, see page 11.

Contributor Information

Bence Hegyi, Email: jbossuyt@ucdavis.edu, Email: bhegyi@ucdavis.edu.

Julie Bossuyt, Email: jbossuyt@ucdavis.edu.

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