Keywords
dilated cardiomyopathy - mitophagy - Kangxian Yixin Granule - PINK1/Parkin pathway
- Chinese medicine - heart failure
Dilated cardiomyopathy (DCM) is a heterogeneous myocardial disease with ventricular
enlargement and reduced myocardial systolic function, which is one of the main causes
of heart failure.[1] The etiology of DCM is diverse and includes genetic and nongenetic causes. At present,
the known genetic causes are mainly related to the mutations of cytoskeletal and sarcomeric
proteins.[2] DCM has a high fatality rate; however, an effective treatment plan for this disease
is lacking, which poses a heavy burden to families and society.[3]
Mitophagy can selectively eliminate impaired or depolarized mitochondria, which is
an important mechanism in the maintenance of cardiac homeostasis.[4] In response to various environmental stresses, dysfunctional mitophagy results in
the accumulation of damaged and dysfunctional mitochondria and excessive mitochondrial
oxidative stress, leading to energy metabolism disorder and a decrease in cardiac
contractile function.[5] A deficiency of mitophagy-related proteins in the heart can directly affect cardiac
function or lead to fatal cardiomyopathy in mouse models.[6]
[7]
[8] Several studies have shown that mitophagy plays a protective role in DCM heart failure.[9] Therefore, restoring the level of mitophagy may be a potential treatment for DCM.
Traditional Chinese medicine has long been used in the treatment of DCM. Invigorating
qi and activating blood circulation is considered a core method in the treatment of
DCM.[10] Kangxian Yixin granule (KXYXG) is an effective clinical treatment for DCM. In our
preliminary clinical study, we confirmed that KXYXG could effectively relieve the
clinical symptoms and improve the left ventricular remodeling and improve the cardiac
function of DCM patients.[11] We were also able to confirm that the therapeutic effect of KXYXG in cases of DCM
is related to reducing myocardial fibrosis and myocardial cell apoptosis and restoring
myocardial energy metabolism in vivo.[12] Hence, this study aimed at exploring the underlying mechanism of the therapeutic
effect of KXYXG on DCM.
Methods
Animals
The α-myosin heavy chain cTnTR141W transgenic mice were purchased from the Institute of Laboratory Animals Science,
Chinese Academy of Medical Sciences and Peking Union Medical College (certificate
no.: 1103261911000021). The mice developed the characteristics of human DCM within
4 months[13]; C57BL/6J mice were purchased from Beijing Charles River Company (certificate no.:
1102861914000028). All mice were raised at the Experimental Animal Center of Henan
Province Hospital of Chinese Medicine at a temperature of (22+2)°C and humidity of
(50± 5)%. This study was approved by the Ethics Committee of Henan Province Hospital
of Chinese Medicine (approval no.: PZ-HNSZYY-2019-004). M-echocardiography was used
to identify the phenotype of the CTNTR141W mice. The phenotype of cardiac dilation was confirmed if the left ventricular end-diastolic
diameter (LVEDD) was >2.64 mm at the age of 2 months.[14]
Drugs
KXYXG contains the following Chinese herbs: Renshen (Ginseng Radix et Rhizoma) 12 g,
Huangqi (Astragali Radix) 30 g, Fuling (Poria) 15 g, Danshen (Salviae Miltiorrhizae
Radix et Rhizoma) 15 g, Cangzhu (Atractylodis Rhizoma) 15 g, Gouqizi (Lycii Fructus)
15 g, Yimucao (Leonuri Herba) 15 g, Maidong (Ophiopogonis Radix) 12 g, Shengma (Cimicifugae
Rhizoma) 9 g. Concentrated granules are prepared from these nine Chinese medicinal
herbs. The batch numbers of the above Chinese herbal formula granules were as follows:
19080058, 20100015, 20040120, 19110126, 20040219, 20100186, 20090042, 20060046, and
20060012. All herbal materials, dispensing granules, and quality control data of KXYXG
were supplied by Sichuan Neo-Green Pharmaceutical Technology Development Co., Ltd
(Sichuan, China). A dose of KXYXG was dissolved in sterile distilled water and heated
to prepare the medicine solution. Coenzyme Q10 (CoQ10) tablets (10 mg/tablet; Eisai
Pharmaceutical Co., Ltd, China, batch no.: 1912001) were crushed, ground, and dissolved
in 50 mL of sterile distilled water to prepare the medicine solution. The solution
was divided into 1.5 mL sterile Eppendorf tubes and stored at −20°C. The dosages of
KXYXG (20.4 g/kg/d) and CoQ10 (1.5 mg/kg/d) were determined according to the equivalent
human dosages.
Groups
Thirty specific pathogen-free (SPF) male cTnTR141W mice with DCM were identified and randomly divided into the model group, KXYXG group,
and CoQ10 group; 10 SPF male C57BL/6J mice formed the normal group. The normal and
model groups were administered normal saline, the KXYXG group was administered the
KXYXG solution, and the CoQ10 group received the CoQ10 solution, once a day, for 8
weeks.
Echocardiography
M-mode echocardiography was performed with an animal echocardiography analysis system
(VEVO1100; VisualSonics Inc., Canada). In brief, the mice were anesthetized by intraperitoneal
injection of 1% pentobarbital sodium solution after weight measurement and fixed in
the supine position; the animals' precardiac region was shaved. M-mode echocardiography
was performed with a 30 MHz transducer. LVEDD and left ventricular end-systolic diameter
(LVESD) were measured, and the left ventricular ejection fraction (EF) and left ventricular
axis shortening rate [fractional shortening (FS)] were calculated by echocardiography.
Hematoxylin and Eosin Staining
Myocardial samples from each group were fixed with 4% paraformaldehyde solution and
embedded in paraffin after decalcification and dehydration. The sections (5 μm) were
stained using hematoxylin and eosin, and morphological characters were observed using
an image autoanalysis system.
Transmission Electron Microscopy
The tissues obtained were cleaned in normal saline at 4°C, and pieces of myocardial
tissues at the apex of the left ventricle were cut and divided into 1 mm3 square samples. The segmented myocardial samples were quickly placed into a centrifugation
tube containing precooled 2.5% glutaraldehyde solution for 4 hours. Thereafter, they
were rinsed with 0.1 mol/L phosphate-buffered saline (PBS) 3 times for 15 minutes
each. Then, 1% osmium was added, stewing for 2 hours. Gradient dehydration was performed
using acetone of different concentrations. The samples were then embedded in epoxy
resin overnight, and gradient curing was performed in the oven at 37°C (12 h), 45°C
(12 h), and 60°C (36 h), after which they were sliced using an ultrathin slicer. Finally,
the samples were dyed with saturated uranium acetate solution and lead citrate solution,
after which transmission electron microscopy was performed.
Measurement of Immunofluorescence Colocalization
Section preparation: Paraffin-embedded sections of the myocardium were prepared as
described previously. The sections were dewaxed, and antigen repair was performed.
Then, after allowing for natural cooling, the slices were placed in PBS solution and
washed 3 times. They were sealed with a 3% (BSA:PTST) solution for 30 minutes, followed
by incubation overnight with a primary antibody at 4°C. The slides were washed and
covered with fluorescent secondary antibody (1:400) drops under dark conditions, and
incubation was performed at 25°C under dark conditions for 1 hour. After that, an
antifade mounting medium with DAPI was dropped onto the slides, and the slides were
incubated at 25 °C for 30 minute in the dark. The sections were observed under a fluorescence
microscope and images were obtained. Fluorescence images were retained using an imaging
system and processed using image-Pro Plus Image analysis software. The position coincidence
of COX4, Parkin, and LC3 was determined, and the Pearson correlation coefficient was
used for quantitative analysis.
Western Blot
Protein was extracted from heart tissue, which was homogenized in RIPA lysis buffer,
and quantitated using the BCA kit (Biyuntian Biological, China). Total protein was
separated on SDS-PAGE gel and then transferred to a PVDF membrane. The membrane was
blocked with 5% skim protein powder at 37°C for 1 hour. The membrane was incubated
with the primary antibody at 4°C overnight. Primary antibodies against p62, PINK1,
Parkin (1:1000 dilution Proteintech, United States), GAPDH (1:2000 dilution Proteintech,
United States), and LC3 (1:1000 dilution Cell Signaling Technology, United States)
were used. After washing, the membrane was treated with a specific horseradish peroxidase–conjugated
secondary antibody (1:2000 dilution Wuhan Sewell Biotechnology, China) for 1.5 hour
and subsequently washed. Relative luminescence intensity was analyzed using a gel
imaging system (Bio-Rad Laboratories, United States).
Statistical Analysis
All data are expressed as means ± standard error of the mean (x̅ ± standard error).
SPSS 22.0 statistical software was used for data analysis. If the measurement data
showed normal distribution and homogeneity of variance, one-way analysis of variance
was used with Tukey's post hoc test. If the data distribution was not normal, a nonparametric
test was used. p < 0.05 was considered statistically significant.
Results
Cardiac Function of Mice Treated with Kangxian Yixin Granule
The cardiac structure and function in DCM mice were measured and calculated using
Vevo1100. As shown in [Fig. 1], the model group showed obvious left ventricular enlargement, left ventricular wall
thinning, decreased ventricular wall movement, decreased systolic function, and a
spherical heart. In comparison with the normal group, the model group showed significantly
higher LVESD and LVEDD and significantly lower FS and LVEF (p < 0.05). In comparison with the corresponding values in the model group, LVESD and
LVEDD significantly decreased after treatment with KXYXG or CoQ10 (p < 0.05), whereas LVEF increased significantly (p < 0.05) and FS was higher (p < 0.05).
Fig. 1 Kangxian Yixin granule (KXYXG) restored cardiac function in DCM mice. Ejection fraction
(EF) values (A). Fractional shortening (FS) values (B). Left ventricular end-diastolic diameter (LVEDD) values (C). Left ventricular end systolic diameter (LVESD) values (D). Representative M-mode echocardiographic images (E). Values are presented as mean ± standard deviation (n = 10 per group); comparison with the control group. ∗
p < 0.05; comparison with the model group, #
p < 0.05.
Myocardial Histopathological Findings of Mice Treated with Kangxian Yixin Granule
As shown in [Fig. 2], the ventriculus cordis of mice in the model group was significantly enlarged and
spherical-shaped and showed a significantly thin ventricle wall. In comparison with
the model group, the KXYXG group showed decreased volume of the left ventricle and
increased relative thickness of the ventricle wall. The reversal effect of KXYXG on
ventricular remodeling was more obvious than that of CoQ10.
Fig. 2 Kangxian Yixin granule (KXYXG) attenuated ventricular remodeling in dilated cardiomyopathy
mice. Representative images of hematoxylin and eosin (×1) and Masson staining (×40).
The cardiomyocytes in the normal group were arranged neatly, with clear stripes and
uniform cytoplasm staining. The nucleus was oval, located in the center of the cell,
and the cells showed no obvious interstitial fiber hyperplasia and edema. In the model
group, the arrangement of cardiomyocytes was relatively disordered and myocardial
tissue was seriously damaged, with irregular hypertrophy of cardiomyocytes and irregular
arrangement of nuclei. In addition, some nuclei were pyrotic and fragmented, and interstitial
edema and interstitial fibrous hyperplasia were observed. In contrast, the KXYXG group
showed a relatively regular arrangement of cardiomyocytes and a relatively clear texture.
Some cardiomyocytes showed irregular hypertrophy, a few cells showed nuclear pyrosis
and fragmentation, and interstitial fiber hyperplasia and edema were not obvious.
In the CoQ10 group, cardiomyocytes were arranged in a relatively regular and clear
texture, and some cardiomyocytes showed irregular hypertrophy.
Myocardial Ultrastructure of Mice Treated with Kangxian Yixin Granule
In comparison with the normal group, the model group showed a significantly altered
ultrastructure of myocardial fibers, with dissolved and broken myocardial myofilaments,
obviously swollen cardiomyocytes, blurred M-line and myofilaments, and destroyed myofibrils.
The mitochondria were disordered and showed irregular and diffuse swelling. However,
the KXYXG and CoQ10 groups showed significantly reduced damage to the myocardial ultrastructure.
Mitochondria in the KXYXG group were mostly round, arranged in an orderly manner,
and significantly greater in number. In the CoQ10 group, some myocardial myofilaments
were dissolved and broken and myocardial cells were swollen. However, the arrangement
of mitochondria was orderly, and most of them were oval-shaped ([Fig. 3]).
Fig. 3 Kangxian Yixin granule (KXYXG) reduced mitochondrial injury in the hearts of dilated
cardiomyopathy mice. Representative images of electronic speculum (×8000).
Immunofluorescence Colocalization of Mice Treated with Kangxian Yixin Granule
Mitochondrial inner membrane COX4 was used to mark the mitochondrial position, and
Parkin and LC3 were used to mark the autophagosome position. DAPI was used to label
the nuclear location. LC3 and COX4 colocalization as well as Parkin and COX4 colocalization
were observed in the same field. Fluorescence images were captured with a camera.
As shown in [Fig. 4A] and [C], in comparison with the normal group, the model group showed no significant difference
in the colocalization of LC3 and COX4 (p > 0.05), with no significant difference. However, in comparison with the model group,
both KXYXG group and CoQ10 group showed a greater degree of colocalization of LC3
and COX4 (p < 0.05). As shown in [Fig. 4B] and [D], in comparison with the normal group, the model group showed reduced degree of colocalization
of Parkin and COX4 (p < 0.05). However, in comparison with the model group, both KXYXG group and CoQ10
group showed greater degree of colocalization of Parkin and COX4 (p < 0.05).
Fig. 4 Kangxian Yixin granule (KXYXG) promoted mitophagy in the hearts of dilated cardiomyopathy
mice. Representative images showing immunofluorescence colocalization of COX4 and
LC3 (A). Representative images showing immunofluorescence colocalization of COX4 and Parkin
(B).The ratio of colocalization of COX4 and LC3 in each group (C). The ratio of colocalization of COX4 and Parkin in each group (D) (n = 6 per group). Comparison with the control group, ∗
p < 0.05; comparison with the model group, #
p < 0.05.
Mitophagy Protein Expression of Mice Treated with Kangxian Yixin Granule
The LC3 II/I ratio is the primary measure for evaluation of autophagy. P62 is a ubiquitin-binding
protein that can target autophagosomes to respond to the elimination of ubiquitinated
proteins. As shown in [Fig. 5A], the LC3 II/I ratio in the myocardial tissue of the model group was lower than that
in the normal group (p > 0.05), with no significant difference. In comparison with the model group, the
KXYXG group showed significantly greater LC3 II/I ratio in the myocardial tissue (p < 0.05), whereas the CoQ10 group only showed a slightly increased LC3 II/I ratio
(p > 0.05). As shown in [Fig. 5B], in comparison with the normal group, the model group showed significantly increased
expression of P62 protein (p < 0.05). However, in comparison with the model group, the KXYXG group and CoQ10 group
showed decreased expressions of the P62 protein (p < 0.05).
Fig. 5 Kangxian Yixin granule (KXYXG) promoted dilated cardiomyopathy heart mitophagy via
PINK1/Parkin pathway. Western blotting was used for the analysis (A–D). Expression of LC3II/LC3I, P62, PINK1, and Parkin protein (E). Comparison with the control group, ∗
p < 0.05; comparison with the model group, #
p < 0.05.
The PINK1/Parkin pathway is a classic pathway of mitophagy. As shown in [Fig. 5C] and [D], in comparison with the normal group, the model group showed reduced expression
of PINK1 protein in the myocardial tissue (p < 0.05). In comparison with the model group, both KXYXG group and CoQ10 group showed
increased PINK1 protein expression (p < 0.05). In comparison with the normal group, the model group showed reduced expression
of the Parkin protein (p < 0.05). However, in comparison with the model group, the KXYXG group showed increased
expression of the Parkin protein (p < 0.05), whereas the CoQ10 group showed slightly increased expression of the same
protein (p > 0.05).
Discussion
DCM, as a myocardial disease defined by pathological features, is mainly diagnosed
based on ventricular dilatation and decreased systolic function of the left ventricle
and exclusion of etiologies of ischemic heart disease, hypertension, and valvular
disease.[15] DCM is currently the most common cause of heart failure worldwide and the most common
indication for heart transplant surgery. Globally, the prevalence of DCM is approximately
40/100,000, and the average annual incidence is approximately 7/100,000.[16] This condition can seriously endanger the health of patients. Recent studies have
suggested that traditional Chinese medicine is effective in the prevention and treatment
of DCM.[17] Some records related to diseases with similar clinical symptoms as DCM have provided
new ideas for the prevention and treatment of DCM. Our team has long been committed
to clinical and basic research on the prevention and treatment of DCM. We believe
that qi deficiency and blood stasis are at the core of the disease, and KXYXG can
be used to treat DCM by invigorating qi and promoting blood circulation. In clinical
studies, KXYXG was found to delay the progression of DCM and improve the cardiac function
of patients.[18] Our previous study found that mice treated with KXYXG for 8 weeks improved significantly
compared with 4 weeks.
With advancements in molecular biology research, the diagnosis rate of familial dilated
cardiomyopathy (FDCM) has also increased.[19] About 30% to 48% of patients with DCM carry DCM-related gene mutations. Moreover,
the pathogenic genes underlying DCM are complex and diverse, with more than 40 genes
reported to be related to DCM.[20] The cardiac troponin T (cTnT) mutation is a common genetic marker in FDCM patients.
The cTnTR141W DCM mouse model we adopted was mainly characterized by severe DCM.[21] In comparison with wild mice, 4-month-old cTnTR141W mice showed obvious pathological manifestations such as whole-heart enlargement,
ventricular wall thinning, reduced cardiac systolic function, and cardiomyocyte hypertrophy,
which were similar to the pathological phenotypes of multiple FDCM patients. Ginsenoside-RB1
and tetramethylpyrazine phosphate have been previously confirmed to improve ventricular
remodeling in the cTnTR141W DCM mouse model, and their mechanism may be closely related to improving myocardial
energy metabolism and inhibiting myocardial fibrosis.[14] In this study, we found that KXYXG could effectively improve cardiac EF in DCM,
postpone ventricular remodeling, and alleviate disorders of myocardial cell arrangement,
interstitial fibrosis, and abnormal myocardial ultrastructure.
Mitochondria are important two-membranous organelles in eukaryotic cells. They play
important roles in cell homeostasis, including the generation of energy through oxidative
phosphorylation, maintenance of calcium homeostasis, and regulation of signals leading
to a programmed cell death. CoQ10 is a commonly used drug for cardiovascular diseases
in clinical conditions[22] and is responsible for transferring electrons from complex I and complex II to complex
III in the mitochondrial respiratory chain, thereby promoting cardiac ATP production.
The results of the Q-SYMBIO trial showed that CoQ10 could significantly improve the
symptoms of heart failure in patients and reduce the occurrence of adverse cardiovascular
events.[23] Therefore, CoQ10 was selected as the control drug in this study. We found that KXYXG
caused a more obvious improvement in ventricular remodeling than CoQ10. Both KXYXG
and CoQ10 had protective effects on DCM myocardial mitochondrial injury. However,
there were significant differences in the mitochondrial morphology between the two
groups after treatment.
Mitophagy is a process in which damaged mitochondria are specifically isolated by
autophagosomes and bound with lysosomes to remove them, and it serves as an important
mechanism of mitochondrial quality control.[24] In general, mitophagy preserves mitochondrial function by removing dysfunctional
mitochondria of the heart.[25] However, both inadequate and aggravated mitophagy can lead to cardiomyopathy. For
example, in diabetic cardiomyopathy, a reduction in mitophagy may contribute to a
beneficial adaptive response to diabetic heart injury.[26] Mitophagy can be divided into receptor-dependent and nonreceptor-dependent categories.
The PINK/Parkin pathway is the most widely studied regulatory pathway for nonreceptor-dependent
mitophagy. Qiliqiangxin improves the symptoms of heart failure by inhibiting the PINK1/Parkin
pathway.[27] The protein expression of PINK1 has also been confirmed to be significantly reduced
in heart failure.[28] ZnCl2 can clear damaged mitochondria by inducing autophagy and improve myocardial ischemia–reperfusion
injury.[29] In this study, we found that KXYXG could improve the fusion of mitochondria and
autophagosomes in DCM mouse myocardial tissue through immunofluorescence colocation.
There is no significant difference in LC3 II/I ratio between the control group and
the model group. As a chronic progressive disease, the changes in the level of autophagy
may be moderate or insignificant in the pathological development of DCM, but after
KXYXG treatment, the level of autophagy is significantly improved. Western blot analysis
confirmed that KXYXG could significantly increase the LC3II/LC3I ratio and decrease
P62 expression in the DCM heart, which was significantly different from the findings
in the CoQ10 group. Simultaneously, the expression level of the PINK1/Parkin protein
significantly increased, indicating that KXYXG could significantly improve the expression
of mitophagy in the DCM heart, thus promoting the clearance of damaged mitochondria
and preventing myocardial injury in DCM.
Conclusion
This study demonstrated that KXYXG has a significant therapeutic effect on DCM and
can inhibit ventricular remodeling and myocardial fibrosis development in DCM mice.
Its therapeutic effect may be to enhance the mitophagy level of myocardial mitochondria
by regulating PINK1/Parkin pathway. This study preliminarily explained the mechanism
of antiventricular remodeling of KXYXG and provided a direction for further research.
