Dapagliflozin treatment and cardiovascular outcome in RBP4/TTRVal30Met (transthyretin cardiac amyloidosis) mice

Abstract

Aims

Whether sodium‐glucose co‐transporter 2 inhibitors are effective for heart failure caused by ATTR‐CA (transthyretin cardiac amyloidosis) remains uncertain. The aim of this study is to investigate the cardiovascular prognosis in ATTR‐CA mice model with dapagliflozin treatment.

Methods and results

Humanized RBP4/TTR^Val50Met and RBP4/TTR mice models were constructed with clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR‐Cas9) techniques and multiple generations breeding. A total of 6 RBP4/TTR mice received placebo treatment, when 12 RBP4/TTR^Val50Met received dapagliflozin (1 mg/kg/day, 6 mice) and placebo (6 mice) treatment. Fasting glucose, intraperitoneal glucose tolerance test, and plasma brain natriuretic peptide (BNP) concentration were measured at Day 0, Week 2, and Week 4. BNP, transforming growth factor‐beta (TGF‐β), collagen type I alpha 1 (COL1A1) protein levels, and Cola1, TGFβ1, TNFα, IL‐1β, BNP relative quantities in cardiac, along with cardiac pathology examination including right ventricular collagen percentage, ventricular septum thickness, left ventricular wall thickness, and left ventricular internal diameter were measured at Week 4 after treatment procedure. All 18 mice completed the experiment. The baseline characteristics were balanced among three treatment groups. In placebo‐treated mice, the cardiac BNP relative quantity was significantly higher in RBP4/TTR^Val50Met mice than RBP4/TTR mice (RBP4[KI/KI], TTR [KI/KI]: 0.72 ± 0.46, RBP4[KI/KI], TTR^Val50Met[KI/KI]: 1.44 ± 0.60, P = 0.043), indicating more significant heart failure progression in ATTR‐CA mice than normal mice. In ATTR‐CA mice, the cardiovascular prognosis measurements including heart failure (plasma BNP concentration and relative quantities of BNP), cardiac inflammation (relative quantities of Cola1, TGFβ1, TNFα, and IL‐1β), and pathological changes (right ventricular collagen percentage, ventricular septum thickness, left ventricular wall thickness, and left ventricular internal diameter) were statistically comparable between those under dapagliflozin and placebo treatment.

Conclusions

Dapagliflozin did not improve cardiovascular prognosis including the progression of heart failure, cardiac inflammation, and pathological changes in ATTR‐CA mice compared with placebo. The results of this study were not in support of dapagliflozin's therapeutic effects for ATTR‐CA. More pre‐clinical and clinical researches to validate these findings and demonstrate the underlying mechanisms are still required.

Introduction

Transthyretin cardiac amyloidosis (ATTR-CA) is a relatively infrequent disease caused by extracellular deposition of transthyretin (TTR)-derived insoluble amyloid fibrils in the myocardium.¹ The major clinical manifestations of ATTR-CA are progressive heart failure with preserved ejection fraction (HFpEF) and restrictive cardiomyopathy.² As the clinical awareness and diagnostic techniques of ATTR-CA have been improved, more patients could be identified and the incidence of ATTR-CA was elevated correspondingly.³ Recent studies indicated that ATTR-CA may account for 13% hospitalized patients with HFpEF⁴ and 16% patients underwent transcatheter aortic valve replacement.⁵

Anomalous TTR deposition holds the chief responsibility in ATTR-CA progression.⁶ TTR is a transporting protein for thyroxine (T4) and retinol binding protein 4 (RBP4). It is mainly produced in liver and brain choroid plexus cells and exists in tetramer form mostly.^7,8 When the normal structure of TTR monomers is changed, it would subsequently facilitate TTR tetramers dissociation, TTR monomers aggregation, and amyloid fibrils formation in myocardial tissues, which leads to ATTR-CA.^6–8 Underlying pathogeneses of this disease might involve TTR gene mutations (known as ATTR variant, ATTRv) or aging-related oxidative stress and cells injury (known as ATTR wild type, ATTRwt).⁹ Current medicine remedies for ATTR-CA primarily refer to TTR tetramer stabilizers (represented by tafamidis) and TTR synthesis silencing agents (small interfering RNA molecules, represented by inotersen and vutrisiran), which were both proved to be effective in postponing ATTR-CA deterioration.^10,11 However, whether regular anti-heart failure agents are effective to ATTR-CA remains unclear.⁶

Sodium-glucose co-transporter 2 (SGLT-2) inhibitors are anti-diabetic drugs that lower blood glucose by restraining urinary glucose reabsorption from inhibiting SGLT-2 proteins in proximal tubule.¹² SGLT-2 inhibitors were demonstrated to reduce the risks of cardiovascular death and hospitalization for heart failure with the potential mechanisms of reducing blood volume through osmotic diuresis, regulating cardiac sympathetic activity, and relieving insulin resistance.^13–15 Although SGLT-2 inhibitors have been recommended as first-line options in treating patients with whole heart failure and HFpEF,¹⁶ evidence for cardiovascular effects of SGLT-2 inhibitors in ATTR-CA is insufficient. A previous real-world study indicated that dapagliflozin was well tolerated in ATTR-CA patients but did not demonstrate the efficacy due to the limitation of the study type.¹⁷ Therefore, with the aim to investigate the therapeutic effects of SGLT-2 inhibitors for ATTR-CA, we designed this study to further investigate the cardiovascular prognosis in ATTRv (RBP4/TTR^Val30Met variant) mice model with dapagliflozin treatment.

Methods

High throughput targets screening

To address whether SGLT-2 inhibitors would introduce cardiovascular benefits for ATTR-CA patients by novel targets beyond regular anti-heart failure mechanisms, we conducted high throughput targets screening and model constructing for dapagliflozin, canagliflozin, and empagliflozin precedingly. The process was carried out through Discovery Studio 2017R2 (DS; BIOVIA-Dassault Systèmes) in a computer with Intel® Xeon® E5-2699 v3 2.30 GHz Octadeca processor core and Windows 8.1 operating system. By targets screening, all three drugs exhibited high affinity to TTR, when dapagliflozin also showed high affinity to RBP4. Detailed outcomes for targets screening and combining models between dapagliflozin and TTR or RBP4 were shown in Supporting Information, Table S1 and Figure S1.

Animal models construction

Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Peking University People's Hospital (No. 2020PHE008). All the procedures were conducted conforming to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.¹⁸ Humanized RBP4/TTR and RBP4/TTR^Val30Met mice model were constructed in Beijing Cyagen Biosciences Co., Ltd (Beijing, China). The animal modelling and breeding procedures were as follows.

Humanized RBP4[KI/KI], TTR[KI/KI], and TTR^Val50Met[KI/KI] mice were constructed as filial generation (F0) initially. According to National Center of Biotechnology Information (NCBI) database, murine RBP4 and TTR genes are located in chromosomes 19 and 18, respectively, and their locus were acquired in line with a previously described method.^19–21 Six exons were identified in murine RBP4, and four exons were recognized in murine TTR. Overview of gene locations and targeting strategies is shown in Supporting Information, Figure S2. To construct humanized RBP4[KI/KI] model, the coding sequence of exon 1 to partial intron 4 sequence was replaced with human RBP4 CDS-rBG pA cassette. To construct humanized TTR[KI/KI] model, the coding sequence of exon 1 plus partial intron 1 sequence was replaced with human TTR CDS-rBG pA cassette. Meanwhile, for humanized TTR^Val50Met[KI/KI] model, the Val50Met (GTG to ATG) mutation site was introduced into human TTR CDS-rBG pA cassette. Homology arms generated by polymerase chain reaction (PCR) using bacterial artificial chromosome (BAC) clone were taken as templates for targeting vectors. Vectors of human RBP4, TTR, or TTR^Val50Met genes with clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease (CRISPR-Cas9) and guide RNA (gRNA) were respectively injected to fertilized ovum of C57BL/6J mice, generating F0. Sequences of human RBP4, TTR, and TTR^Val50Met genes and corresponding gRNA are exhibited in Supporting Information, Table S2.

F0 mice were hybridized with wild-type (WT) C57BL/6J mice to produce humanized RBP4[KI/+], TTR[KI/+], and TTR^Val50Met[KI/+] mice model as F1. Through multiple generations of interbreeding from F1, mice with genotypes of (RBP4[KI/KI], TTR[KI/KI]) or (RBP4[KI/KI], TTR^Val50Met[KI/KI]) were produced. Reverse transcription-polymerase chain reaction (RT-PCR) with agarose gel electrophoresis (AGE) (specific primers are summarized in Supporting Information, Table S3) and southern blots analysis of the RT-PCR products were correspondingly performed to identify the genotypes of mice models. All mice models were proved to meet the anticipated genotypes (Supporting Information, Figures S3 and S4).

All the mice involved in this study were housed in cages (three to five mice per cage) and were placed in a room at constant temperature of 26°C with 12:12 h light–dark period. All the mice were with unlimited access to water and fodders.

Animal treatment

Six mice of (RBP4[KI/KI], TTR[KI/KI]) genotype and 12 mice of (RBP4[KI/KI], TTR^Val50Met[KI/KI]) genotype with the age beyond 10 weeks were included in this study. They were randomly divided into three parallel treatment groups with six in each as the following: (i) (RBP4[KI/KI], TTR[KI/KI]) genotype receiving placebo gavage; (ii) (RBP4[KI/KI], TTR^Val50Met[KI/KI]) genotype receiving dapagliflozin gavage; and (iii) (RBP4[KI/KI], TTR^Val50Met[KI/KI]) genotype receiving placebo gavage. All the mice received either 1 mg/kg/day of dapagliflozin (dissolved in normal saline and configured as solutions with concentration of 0.1 mg/1 mL) (AstraZeneca Pharmaceuticals Co., Ltd, USA) or 10 mL/kg/day of placebo (normal saline) for 4 weeks according to their groups. Fasting glucose, weight, and fodder intake of each mouse were measured at baseline, Day 0 (D0), Week 2, and Week 4. At the end of treatment, all the mice were sacrificed by cervical dislocation disposal. The cardiac and aorta tissues were immediately excised with cold storage thereafter.

Glucose tolerance test

Intraperitoneal glucose tolerance test (IPGTT) was conducted at D0, Week 2, and Week 4. All the mice should be fast for 12 h before IPGTT. During IPGTT, 5% glucose (10 mL/kg) was injected intraperitoneally, and blood samples were collected from tail veins before (0 min) and after (15, 30, 60, 90, and 120 min) glucose administration. Blood glucose was measured with automatic glucometer (Yuwell Medical Equipment Co., Ltd, China).

Western blots

Western blots were implemented to measure the contents of brain natriuretic peptide (BNP), transforming growth factor-beta (TGF-β), and collagen type I alpha 1 (COL1A1) in mice cardiac tissues after treatment. A fraction of the frozen cardiac tissues (50 mg) was cut and homogenized in lysis buffer (mixture of Tris–HCl pH 8.0, 50 mmol/L of sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and protease inhibitors). The tissue suspensions were centrifuged at 12 000 rpm, 4°C for 15 min, and the supernatants were collected. Protein concentration was determined by bicinchoninic acid (BCA) protein assay kits (Beyotime Biotechnology Co., Ltd, China). Supernatants were mixed with loading buffer (mixture of Tris–HCl pH 8.0, sodium dodecyl sulfate, 17.4% glycerol, 0.026% bromophenol blue, and 8.7% β-mercaptoethanol) and were boiled for 5 min. Equal amount of 50 μg of protein was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12% polyacrylamide gels) and was transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Trading Co., Ltd, USA). Membranes were incubated with primary antibodies of BNP (Affinity Biosciences Co., Ltd, Australia), TGF-β (Huaan Bioscience Co., Ltd, China), COL1A1 (Abcam Trading Co., Ltd, UK), β-tubulin (Beijing Solarbio Science & Technology Co., Ltd, China), and secondary antibodies as horseradish peroxidase (HRP) conjugated antibodies (Beyotime Biotechnology Co., Ltd). The protein contents were measured with grey-scale scanning software (ImageJ 1.48, NIH) and were rectified by β-tubulin.

Enzyme-linked immunosorbent assays (ELISA)

ELISA at D0, Week 2, and Week 4 were performed to identify the serum BNP concentrations of all the mice after treatment (mouse BNP kit, Wuhan Hualian Biotechnology Co., Ltd, China). All the procedures were carried out following the specifications of the manufacturer.

Real-time quantitative polymerase chain reaction (RT-qPCR)

RT-qPCR was conducted to determine the expression quantity of Cola1, TGFβ1, TNFα, IL-1β, and BNP in mice cardiac tissues after treatment. Operators extracted RNA samples from cardiac tissues (50 mg) by Trizol (Invitrogen Trading Co., Ltd, USA) according to the manufacturer's protocols. RNA samples were then reversely transcribed into cDNA and then into RT-qPCR amplification (Roche LightCycler® 480II, Roche Trading Co., Ltd, Switzerland). Specific primers for Cola1, TGFβ1, TNFα, IL-1β, BNP, and GAPDH (housekeeping gene) were designed to identify the mRNA levels (Table 2). A melting curve analysis was conducted to validate the specificity of RT-qPCR products. CT (cycle threshold) was the minimum cycle turns for the nucleic acid fluorescence signal to be detectable. The expression quantities of the above genes were rectified with GAPDH as relative quantities by 2^−ΔΔCT methods.

Cardiac histological pathology

Mice cardiac histological pathology examination was performed to evaluate the cardiac lesions by right ventricular collagen percentage, ventricular septum thickness, left ventricular wall thickness, and left ventricular internal diameter after treatment. After the sampling procedures for the above mentioned items, cardiac tissues were fixed and embedded in paraffin. In analysis for right ventricular collagen percentage, cardiac sections of 4 μm were stained with Masson's trichrome. Fibrosis areas and their proportions were measured (3 fields for each mouse, magnification of ×200 with a microscope) with Image-Pro Plus 6.0 (Media Cybernetics, Co., Ltd, Rockville, MD, USA). In analysis for ventricular septum thickness, left ventricular wall thickness, and left ventricular internal diameter, longitudinal sections for cardiac were stained with haematoxylin and eosin. Thicknesses and diameters (5 sections for each mouse, magnification of ×50 with a microscope) were also measured with Image-Pro Plus 6.0 (Media Cybernetics, Co., Ltd).

Statistical analysis

Data were presented in the format of mean ± standard deviation (SD). Statistical analyses were performed with IBM SPSS Version 26.0. Statistical difference between two groups was examined by Student's t-test. The analysis of variance (ANOVA) and Bonferroni's test were applied for the comparison between more than two groups. Statistical significance was considered at P < 0.05.

Results

From the inception, a total of 18 mice were included in this study and were divided into three groups according to different genotypes and treatment drugs. Baseline characteristics of all the mice are summarized in Table 1. No significant difference was found in terms of baseline body weight (P = 0.29) or fasting glucose level (P = 0.30) among these three groups.

Table 1 Baseline characteristics of the 18 mice that completed the treatment

Genotypes	(RBP4[KI/KI], TTR[KI/KI])	(RBP4[KI/KI], TTRVal50Met[KI/KI])
Treatment	Placebo	Dapagliflozin (1 mg/kg/day)	Placebo
Numbers	6	6	6
Weight (g)	24.45 ± 3.16	26.67 ± 2.16	25.23 ± 1.48
Fasting glucose (mmol/L)	7.40 ± 1.72	7.85 ± 1.63	6.57 ± 0.50

Fasting glucose and intraperitoneal glucose tolerance test

In (RBP4[KI/KI], TTR^Val50Met[KI/KI]) genotype mice, no significant difference was found in the area under the curve (AUC) of the IPGTT between dapagliflozin treatment and placebo. Moreover, between (RBP4[KI/KI], TTR^Val50Met[KI/KI]) and (RBP4[KI/KI], TTR[KI/KI]) mice with placebo treatment, no significant difference was shown in glucose AUC of IPGTT as well (Figure 1 and Table 2).

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Table 2 Summary of the results and Student's t-test

Outcome measurements	Dapagliflozin treatment group (mean ± SD)	Placebo treatment group (mean ± SD)	P values for t-test
Dapagliflozin treatment vs. placebo treatment in mice with (RBP4[KI/KI], TTRVal50Met[KI/KI]) genotype
Weight D0 (g)	26.67 ± 2.16	25.23 ± 1.48	0.21
Weight Week 1	28.53 ± 2.11	27.10 ± 2.56	0.32
Weight Week 2	28.35 ± 3.03	27.40 ± 2.89	0.59
Weight Week 3	29.30 ± 2.19	27.58 ± 2.37	0.22
Weight Week 4	26.97 ± 1.52	25.50 ± 2.10	0.20
Weight change from baseline (Week 2)	1.68 ± 1.47	2.17 ± 2.70	0.71
Weight change from baseline (Week 4)	0.30 ± 1.73	0.27 ± 1.72	0.97
Fasting blood glucose D0 (mmol/L)	7.85 ± 1.63	6.57 ± 0.50	0.10
Fasting blood glucose Week 2	5.85 ± 1.40	6.47 ± 0.88	0.38
Fasting blood glucose Week 4	5.27 ± 1.13	6.77 ± 1.51	0.080
IPGTT AUC D0 (mmol/L * min)	1700.50 ± 263.01	1603.75 ± 109.88	0.43
IPGTT AUC Week 2	1676.13 ± 325.16	1402.75 ± 281.87	0.15
IPGTT AUC Week 4	1633.38 ± 283.05	1773.88 ± 338.76	0.45
Wb BNP/β-tubulin	1.21 ± 0.29	1.16 ± 0.40	0.81
Wb TGF-β/β-tubulin	0.88 ± 0.11	0.90 ± 0.52	0.93
Wb COL1A1/β-tubulin	0.33 ± 0.17	0.66 ± 0.63	0.24
Cola1 relative quantity	1.14 ± 0.80	1.49 ± 1.70	0.66
TGFβ1 relative quantity	1.40 ± 0.49	1.09 ± 0.38	0.26
TNFα relative quantity	0.23 ± 0.14	0.32 ± 0.13	0.28
IL-1β relative quantity	0.36 ± 0.20	0.38 ± 0.12	0.79
BNP relative quantity	1.87 ± 0.74	1.44 ± 0.60	0.29
ELISA BNP concentration D0 (ng/mL)	70.04 ± 6.78	76.61 ± 1.98	0.046
ELISA BNP concentration Week 2	59.88 ± 5.15	64.19 ± 5.26	0.18
ELISA BNP concentration Week 4	64.17 ± 3.18	68.09 ± 5.07	0.14
Right ventricular collagen percentage (%)	0.032 ± 0.023	0.022 ± 0.015	0.24
Ventricular septum thickness (μm)	1325.75 ± 420.63	800.96 ± 154.01	0.20
Left ventricular wall thickness (μm)	879.10 ± 153.33	951.43 ± 221.90	0.69
Left ventricular internal diameter (μm)	2009.19 ± 347.02	1773.54 ± 151.66	0.45
Outcome measurements	(RBP4[KI/KI], TTR[KI/KI]) group	(RBP4[KI/KI], TTRVal50Met[KI/KI]) group	P values
(RBP4[KI/KI], TTR[KI/KI]) vs. (RBP4[KI/KI], TTRVal50Met[KI/KI]) in mice treated with placebo
Weight D0 (g)	24.45 ± 3.16	25.23 ± 1.48	0.60
Weight Week 1	26.23 ± 2.78	27.10 ± 2.56	0.59
Weight Week 2	25.75 ± 2.39	27.40 ± 2.89	0.31
Weight Week 3	25.68 ± 2.16	27.58 ± 2.37	0.18
Weight Week 4	24.30 ± 2.89	25.50 ± 2.10	0.43
Weight change from baseline (Week 2)	1.30 ± 1.58	2.17 ± 2.70	0.51
Weight change from baseline (Week 4)	−0.15 ± 1.53	0.27 ± 1.72	0.67
Fasting blood glucose D0 (mmol/L)	7.40 ± 1.72	6.57 ± 0.50	0.28
Fasting blood glucose Week 2	7.25 ± 1.98	6.47 ± 0.88	0.40
Fasting blood glucose Week 4	7.08 ± 1.33	6.77 ± 1.51	0.71
IPGTT AUC D0 (mmol/L * min)	1785.00 ± 373.89	1603.75 ± 109.88	0.28
IPGTT AUC Week 2	1641.38 ± 296.52	1402.75 ± 281.87	0.18
IPGTT AUC Week 4	2174.50 ± 344.85	1773.88 ± 338.76	0.07
Wb BNP/β-tubulin	0.90 ± 0.23	1.16 ± 0.40	0.21
Wb TGF-β/β-tubulin	0.83 ± 0.13	0.90 ± 0.52	0.74
Wb COL1A1/β-tubulin	0.45 ± 0.37	0.66 ± 0.63	0.50
Cola1 relative quantity	1.47 ± 0.86	1.49 ± 1.70	0.98
TGFβ1 relative quantity	1.10 ± 0.38	1.09 ± 0.38	0.97
TNFα relative quantity	0.35 ± 0.25	0.32 ± 0.13	0.83
IL-1β relative quantity	0.41 ± 0.18	0.38 ± 0.12	0.76
BNP relative quantity	0.72 ± 0.46	1.44 ± 0.60	0.043
ELISA BNP concentration D0 (ng/mL)	74.74 ± 4.42	76.61 ± 1.98	0.37
ELISA BNP concentration Week 2	66.12 ± 3.91	64.19 ± 5.26	0.49
ELISA BNP concentration Week 4	70.01 ± 3.04	68.09 ± 5.07	0.44
Right ventricular collagen percentage (%)	0.044 ± 0.039	0.022 ± 0.015	0.24
Ventricular septum thickness (μm)	906.85 ± 324.15	800.96 ± 154.01	0.72
Left ventricular wall thickness (μm)	1013.48 ± 69.83	951.43 ± 221.90	0.74
Left ventricular internal diameter (μm)	1546.00 ± 816.62	1773.54 ± 151.66	0.74

Plasma BNP concentrations

In those with genotypes of (RBP4[KI/KI], TTR^Val50Met[KI/KI]), after treatment of 2 and 4 weeks, the plasma BNP concentrations were comparable between dapagliflozin treatment and placebo. Between those with (RBP4[KI/KI], TTR[KI/KI]) and (RBP4[KI/KI], TTR^Val50Met[KI/KI]) genotypes, no significant difference was observed after placebo treatment (Table 2).

Relative quantities of Cola1, TGFβ1, TNFα, IL-1β, BNP, and protein levels of BNP, TGF-β, COL1A1 in the cardiac of mice

With placebo treatment, those with (RBP4[KI/KI], TTR^Val50Met[KI/KI]) genotype exhibited significantly higher BNP relative quantity than those with (RBP4[KI/KI], TTR[KI/KI]) genotype (RBP4[KI/KI], TTR [KI/KI]: 0.72 ± 0.46, RBP4[KI/KI], TTR^Val50Met[KI/KI]: 1.44 ± 0.60, P = 0.043). No significant difference was found in the relative quantity of Cola1, TGFβ1, TNFα, or IL-1β among all experimental groups (Figure 2 and Table 2). In addition, no significant difference was observed in the protein level of BNP, TGF-β, or COL1A1 in the cardiac of mice among all experimental groups (Supporting Information, Figure S5 and Table 2).

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Cardiac histological pathology

The histological pathology inspections were implemented to determine the right ventricular collagen percentage, ventricular septum thickness, left ventricular wall thickness, and left ventricular internal diameter for the cardiac of mice. However, no significance was found (Figure 3 and Table 2).

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Discussion

To our knowledge, this is the first animal experiment research investigating the cardiovascular prognosis of ATTR-CA mice with dapagliflozin treatment. Humanized RBP4/TTR and RBP4/TTR^Val50Met mice models were established for the research aims. According to study results, the relative quantity of BNP in cardiac was significantly higher in RBP4/TTR^Val50Met mice than RBP4/TTR mice in placebo treatment groups, suggesting the heart failure phenotype of RBP4/TTR^Val50Met. Nevertheless, dapagliflozin did not improve cardiovascular prognosis items including the degrees of heart failure, cardiac inflammation, and pathological changes in ATTR-CA mice compared with placebo. The outcomes of this research were not in support of the assumption that dapagliflozin has remedying effects for ATTR-CA.

ATTR-CA is a myocardial disease caused by aberrant TTR deposition and subsequent amyloid fibrils formation in myocardium tissues.^1–3 Previously, ATTR-CA was considered as a rare condition with estimated incidence of only 50 000 worldwide.²⁰ While attributing to the evolution of cardiac non-biopsy diagnostic techniques, the diagnostic rates of ATTR-CA have increased and it has become a condition that every cardiologist could encounter. It was suggested that over 10% of the population over 80 years old were with TTR-related amyloid deposition in cardiac and other organs.²¹

The pathological changes of ATTR-CA principally involved increased amyloid fibrils deposition in myocardium, symmetric ventricular wall and septum incrassation, and reduced ventricular diameters.²² The major clinical manifestations of ATTR-CA were progressive HFpEF and restrictive cardiomyopathy with decreased diastolic function (owing to reduced myocardial compliance).⁶ As TTR deposition continued, the severity of heart failure would aggravate and develop into heart failure with reduced ejection fraction (HFrEF).²³ Other symptoms concerning arrhythmia, conduction blocking, and multiple organs involvement (mainly renal and central/periphery nerves) might occur.¹¹ The progression of ATTR-CA was reported severe and rapid, with the median survival time of only 3.5–5 years for ATTRwt and 4–5 years for ATTRv.^24,25 Correspondingly in this research, we identified a significantly higher BNP relative quantity in ATTRv mice model than normal RBP4/TTR mice model under placebo treatment, indicating the progression of heart failure in mice with ATTR-CA compared with normal ones. For treatment of ATTR-CA, apart from aetiological therapies (including tafamidis and so on), anti-heart failure therapies also serve as important means of remedy for ATTR-CA in whole periods.^26–28

As emerging hypoglycaemic drugs with anti-heart failure ability, SGLT-2 inhibitors were considered to be with therapeutic effects for ATTR-CA as well. In addition to the previously mentioned regular anti-heart failure mechanisms,^13–15 SGLT-2 inhibitors were also demonstrated able to alleviate HFpEF by activating cyclic guanosine monophosphate (cGMP) pathways to improve myocardial compliance, acting on adenosine monophosphate activated protein kinase (AMPK) pathways to inhibit fibrosis and myocardial remodelling, and modulating intracellular Ca²⁺-Na⁺ homeostasis to lower myocardial tone.^29–32 Though dapagliflozin was found not effective in ameliorating heart failure phenotypes in high-fat diet plus angiotensin II (ANG II)-induced HFpEF mice model,³³ several large randomized controlled trials (RCTs) indicated that SGLT-2 inhibitors are effective in improving cardiovascular prognosis of HFpEF patients.^34,35 Moreover, according to the high throughput targets screening process we implemented, dapagliflozin showed high affinity to both TTR and RBP4 (Table S1). TTR serves as the main pathogenic factor of ATTR-CA, while RBP4 was demonstrated to be cardiovascular pathogenic by inducing interleukin-6 (IL-6)-related cardiovascular inflammation, insulin resistance, and cholesterol infiltration.^36–38 As ATTR-CA is characterized with HFpEF and restrictive cardiomyopathy, and dapagliflozin was presumed to act on therapeutic targets of TTR or RBP4 and inhibit their pathogenicity, SGLT-2 inhibitors might be promising in ATTR-CA treatment.

However, in this research, it was revealed that the cardiovascular prognoses (including the degrees of heart failure, cardiac inflammation, and pathological changes progression) in ATTR-CA mice with dapagliflozin treatment were comparable to mice with placebo treatment, indicating non-significant therapeutic effects of dapagliflozin for ATTR-CA. There were also no antecedent pre-clinical or clinical researches demonstrating the effects of SGLT-2 inhibitors (especially dapagliflozin) for ATTR-CA. As there may be discrepancies between pre-clinical evidence and clinical research outcomes, more studies exploring the efficacy of SGLT-2 inhibitors for ATTR-CA should be implemented to provide further sufficient evidence.

There are some limitations in this research. First of all, the experimental periods of this study were only 4 weeks, which might not be able to adequately evaluate the cardiovascular effects of dapagliflozin or fully establish the ATTR-CA phenotype in mice. Meanwhile, more precise evaluation measurements for heart failure including ultrasound cardiogram and cardiac angiography were not conducted due to the lack of facilities. As the first animal experiment research in this field, our study replenished evidence for dapagliflozin in the treatment of ATTR-CA and provided new insights for repurposing SGLT-2 inhibitors to this disease. Though results of this study exhibited non-significant therapeutic effects of dapagliflozin for ATTR-CA, further researches to validate these findings and explore the underlying mechanisms, cardiovascular benefits, and efficacy and safety for SGLT-2 inhibitors in ATTR-CA patients are still warranted.

Conclusions

In this research, we accomplished the construction of humanized RBP4/TTR (normal genotype) and RBP4/TTR^Val50Met (ATTRv genotype) mice model. It was found that RBP4/TTR^Val50Met mice were with significantly higher BNP relative quantity than RBP4/TTR mice. However, the cardiovascular prognosis parameters including heart failure parameters, cardiac inflammation measurements, and pathological changes were comparable between dapagliflozin-treated and placebo-treated RBP4/TTR^Val50Met mice. The results were not in support of the therapeutic effects of dapagliflozin for ATTR-CA. More researches to validate these findings and to demonstrate the underlying mechanisms are still required.

Conflict of interest

L.J. has received fees for lecture presentations and for consulting from AstraZeneca, Merck, Metabasis, MSD, Novartis, Eli Lilly, Roche, Sanofi-Aventis, and Takeda. All authors have completed the ICMJE uniform disclosure form at (available on request from the corresponding authors) and declare no other support from any organization for the submitted work other than that described above.

Funding

This work was supported by the National Natural Science Foundation of China (No. 81970698) and the Natural Science Foundation of Beijing Municipality (No. 7202216). The funding agencies had no roles in the study design, data collection or analysis, decision to publish, or preparation of the manuscript.

References

Di Lisi D, Di Stefano V, Brighina F, Galassi AR, Novo G. Therapy of ATTR cardiac amyloidosis: Current indications. Curr Probl Cardiol 2023;48: [eLocator: 101487]. doi:10.1016/j.cpcardiol.2022.101487

Yamamoto H, Yokochi T. Transthyretin cardiac amyloidosis: An update on diagnosis and treatment. ESC Heart Fail 2019;6:1128–1139. doi:10.1002/ehf2.12518

Obi CA, Mostertz WC, Griffin JM, Judge DP. ATTR epidemiology, genetics, and prognostic factors. Methodist Debakey Cardiovasc J 2022;18:17–26. doi:10.14797/mdcvj.1066

Gonzalez‐Lopez E, Gallego‐Delgado M, Guzzo‐Merello G. Wild‐type transthyretin amyloidosis as a cause of heart failure with preserved ejection fraction. Eur Heart J 2015;36:2585–2594. doi:10.1093/eurheartj/ehv338

Koike H, Katsuno M. Transthyretin amyloidosis: Update on the clinical spectrum, pathogenesis, and disease‐modifying therapies. Neurol Ther 2020;9:317–333. doi:10.1007/s40120‐020‐00210‐7

Griffin JM, Rosenthal JL, Grodin JL, Maurer MS, Grogan M, Cheng RK. ATTR amyloidosis: Current and emerging management strategies: JACC: CardioOncology state‐of‐the‐art review. JACC CardioOncol 2021;3:488–505. doi:10.1016/j.jaccao.2021.06.006

Ueda M. Transthyretin: Its function and amyloid formation. Neurochem Int 2022;155: [eLocator: 105313]. doi:10.1016/j.neuint.2022.105313

Vieira M, Saraiva MJ. Transthyretin: A multifaceted protein. Biomol Concepts 2014;5:45–54. doi:10.1515/bmc‐2013‐0038

Porcari A, Merlo M, Rapezzi C, Sinagra G. Transthyretin amyloid cardiomyopathy: An uncharted territory awaiting discovery. Eur J Intern Med 2020;82:7–15. doi:10.1016/j.ejim.2020.09.025

Nativi‐Nicolau J, Judge DP, Hoffman JE, Gundapaneni B, Keohane D, Sultan MB, et al. Natural history and progression of transthyretin amyloid cardiomyopathy: Insights from ATTR‐ACT. ESC Heart Fail 2021;8:3875–3884. doi:10.1002/ehf2.13541

Benbrahim M, Norman K, Sanchorawala V, Siddiqi OK, Hughes D. A review of novel agents and clinical considerations in patients with ATTR cardiac amyloidosis. J Cardiovasc Pharmacol 2021;77:544–548. doi:10.1097/FJC.0000000000001004

Clar C, Gill JA, Court R, Waugh N. Systematic review of SGLT2 receptor inhibitors in dual or triple therapy in type 2 diabetes. BMJ Open 2012;2: [eLocator: e001007]. doi:10.1136/bmjopen‐2012‐001007

Lambers Heerspink HJ, de Zeeuw D, Wie L, Leslie B, List J. Dapagliflozin a glucose‐regulating drug with diuretic properties in subjects with type 2 diabetes. Diabetes Obes Metab 2013;15:853–862. doi:10.1111/dom.12127

Jia G, Whaley‐Connell A, Sowers JR. Diabetic cardiomyopathy: A hyperglycaemia‐ and insulin‐resistance‐induced heart disease. Diabetologia 2018;61:21–28. doi:10.1007/s00125‐017‐4390‐4

Rahman A, Fujisawa Y, Nakano D, Hitomi H, Nishiyama A. Effect of a selective SGLT2 inhibitor, luseogliflozin, on circadian rhythm of sympathetic nervous function and locomotor activities in metabolic syndrome rats. Clin Exp Pharmacol Physiol 2017;44:522–525. doi:10.1111/1440‐1681.12725

McDonagh TA, Metra M, Adamo M, Gardner RS, Baumbach A, Böhm M, et al. 2021 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure. Eur Heart J 2021;42:3599–3726. doi:10.1093/eurheartj/ehab368

Dobner S, Bernhard B, Asatryan B, Windecker S, Stortecky S, Pilgrim T, et al. SGLT2 inhibitor therapy for transthyretin amyloid cardiomyopathy: Early tolerance and clinical response to dapagliflozin. ESC Heart Fail 2023;10:397–404. doi:10.1002/ehf2.14188

National Research Council (US) Committee for the Update of the Guide for the Care and Use of Laboratory Animals. Guide for the Care and Use of Laboratory Animals. 8th ed. Washington (DC): National Academies Press (US); 2011.

Li X, Lyu Y, Shen J, Mu Y, Qiang L, Liu L, et al. Amyloid deposition in a mouse model humanized at the transthyretin and retinol‐binding protein 4 loci. Lab Invest 2018;98:512–524. doi:10.1038/s41374‐017‐0019‐y

Gertz MA. Hereditary ATTR amyloidosis: Burden of illness and diagnostic challenges. Am J Manag Care 2017;23:S107–S112. doi:10.1002/msj.21352

Cornwell GG 3rd, Murdoch WL, Kyle RA, Westermark P, Pitkänen P. Frequency and distribution of senile cardiovascular amyloid. A clinicopathologic correlation. Am J Med 1983;75:618–623. doi:10.1016/0002‐9343(83)90443‐6

Maurer MS, Elliott P, Comenzo R, Semigran M, Rapezzi C. Addressing common questions encountered in the diagnosis and management of cardiac amyloidosis. Circulation 2017;135:1357–1377. doi:10.1161/CIRCULATIONAHA.116.024438

Sekijima Y. Transthyretin (ATTR) amyloidosis: Clinical spectrum, molecular pathogenesis and disease‐modifying treatments. J Neurol Neurosurg Psychiatry 2015;86:1036–1043. doi:10.1136/jnnp‐2014‐308724

Rapezzi C, Merlini G, Quarta CC, Riva L, Longhi S, Leone O, et al. Systemic cardiac amyloidoses: Disease profiles and clinical courses of the 3 main types. Circulation 2009;120:1203–1212. doi:10.1161/CIRCULATIONAHA.108.843334

Fontana M, Ćorović A, Scully P, Moon JC. Myocardial amyloidosis: The exemplar interstitial disease. JACC Cardiovasc Imaging 2019;12:2345–2356. doi:10.1016/j.jcmg.2019.06.023

Emdin M, Aimo A, Rapezzi C, Fontana M, Perfetto F, Seferović PM, et al. Treatment of cardiac transthyretin amyloidosis: An update. Eur Heart J 2019;40:3699–3706. doi:10.1093/eurheartj/ehz298

Barge‐Caballero G, Barge‐Caballero E, López‐Pérez M, Bilbao‐Quesada R, González‐Babarro E, Gómez‐Otero I, et al. Beta‐blocker exposure and survival in patients with transthyretin amyloid cardiomyopathy. Mayo Clin Proc 2022;97:261–273. doi:10.1016/j.mayocp.2021.08.006

Pollak A, Falk RH. Left ventricular systolic dysfunction precipitated by verapamil in cardiac amyloidosis. Chest 1993;104:618–620. doi:10.1378/chest.104.2.618

Buckley LF, Shah AM. Recent advances in the treatment of chronic heart failure. F1000Res 2019;8:F1000 Faculty Rev‐2134:2134. doi:10.12688/f1000research.20447.1

Pabel S, Hamdani N, Singh J, Sossalla S. Potential mechanisms of SGLT2 inhibitors for the treatment of heart failure with preserved ejection fraction. Front Physiol 2021;12: [eLocator: 752370]. doi:10.3389/fphys.2021.752370

Ye Y, Jia X, Bajaj M, Birnbaum Y. Dapagliflozin attenuates Na⁺/H⁺ exchanger‐1 in cardiofibroblasts via AMPK activation. Cardiovasc Drugs Ther 2018;32:553–558. doi:10.1007/s10557‐018‐6837‐3

Xue M, Li T, Wang Y, Chang Y, Cheng Y, Lu Y, et al. Empagliflozin prevents cardiomyopathy via sGC‐cGMP‐PKG pathway in type 2 diabetes mice. Clin Sci (Lond) 2019;133:1705–1720. doi:10.1042/CS20190585

Withaar C, Meems LMG, Markousis‐Mavrogenis G, Boogerd CJ, Silljé HHW, Schouten EM, et al. The effects of liraglutide and dapagliflozin on cardiac function and structure in a multi‐hit mouse model of heart failure with preserved ejection fraction. Cardiovasc Res 2021;117:2108–2124. doi:10.1093/cvr/cvaa256

Packer M, Butler J, Zannad F, Filippatos G, Ferreira JP, Pocock SJ, et al. Effect of empagliflozin on worsening heart failure events in patients with heart failure and preserved ejection fraction: EMPEROR‐Preserved trial. Circulation 2021;144:1284–1294. doi:10.1161/CIRCULATIONAHA.121.056824

Nassif ME, Windsor SL, Borlaug BA, Kitzman DW, Shah SJ, Tang F, et al. The SGLT2 inhibitor dapagliflozin in heart failure with preserved ejection fraction: A multicenter randomized trial. Nat Med 2021;27:1954–1960. doi:10.1038/s41591‐021‐01536‐x

Majerczyk M, Olszanecka‐Glinianowicz M, Puzianowska‐Kuźnicka M, Chudek J. Retinol‐binding protein 4 (RBP4) as the causative factor and marker of vascular injury related to insulin resistance. Postepy Hig Med Dosw (Online) 2016;70:1267–1275. doi:10.5604/17322693.1226695

Liu Y, Zhong Y, Chen H, Wang D, Wang M, Ou JS, et al. Retinol‐binding protein‐dependent cholesterol uptake regulates macrophage foam cell formation and promotes atherosclerosis. Circulation 2017;135:1339–1354. doi:10.1161/CIRCULATIONAHA.116.024503

Steinhoff JS, Lass A, Schupp M. Biological functions of RBP4 and its relevance for human diseases. Front Physiol 2021;12: [eLocator: 659977]. doi:10.3389/fphys.2021.659977