Tongmai formula improves cardiac function via regulating mitochondrial quality control in the myocardium with ischemia/reperfusion injury

https://doi.org/10.1016/j.biopha.2020.110897Get rights and content
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Highlights

  • Tongmai formula improved cardiac mitochondrial function during myocardial ischemia/reperfusion injury of rats.

  • Mitochondrial morphology of neonatal rat left ventricular myocytes was analyzed based on an image algorithm.

  • Tongmai formula regulated mitochondrial dynamics of hypoxia/reoxygenation injured neonatal rat left ventricular myocytes.

Abstract

Background

Mitochondrial quality control, regulated by mitochondrial dynamics and mitophagy, has been regarded as pivotal process to induce segregation of mitochondria during myocardial ischemia/reperfusion (I/R) injury. However, few works revealed the regulation of mitochondrial quality control by therapeutic agents. Tongmai formula (TM) is a clinically used botanical drug for treating cardiovascular diseases, which mechanism is unveiled. Thus, in this study, we investigated the pharmacological effects of TM on modulating mitochondrial quality control during cardiac injury.

Methods

Rats subjected to myocardial I/R injury and neonatal rat ventricular myocytes (NRVMs) exposed to hypoxia/reoxygenation (H/R) were used to simulate cardiac injury during myocardial ischemia/reperfusion process. Morphological examination, histopathological examination, echocardiography, and immunohistochemistry were used to determine the cardiac injury after I/R injury. Biochemical indices in serum were estimated by the enzyme-linked immunosorbent assays (ELISA). 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) was used for mitochondrial membrane potential (ΔΨm) evaluation. 2′,7′-dichlorofluorescin diacetate (DCFH-DA) was used for intracellular reactive oxygen species (ROS) evaluation. Mitochondria in NRVMs were labeled by tetramethylrhodamine methyl ester (TMRM) for mitochondrial morphosis imaging and estimation. Western blotting was used for cytochrome c (CYCS), apoptosis inducing factor (AIF) and mitofusin 2 (Mfn2) contents evaluation. Immunochemistry fluorescence was used for dynamin related protein 1 (Drp1) expression measurement.

Results

TM treatment markedly decreased myocardium infarct size. It also significantly improved left ventricular contractile function and alleviated cardiomyocytes apoptosis, as well as reduced the production of cardiac troponin T, creatine kinase, lactate dehydrogenase, malondialdehyde and elevated glutathione and superoxide dismutase. Intriguingly, we found that mitochondrial membrane potential loss and mitochondrial permeability transition pore (mPTP) opening were recovered after TM treatment. It also down-regulated cytochrome c and apoptosis inducing factor contents after myocardial I/R injury. In vitro study showed that TM treatment reduced intracellular ROS content and recovered ΔΨm in NRVMs after H/R injury. We also observed that TM could reduce the expression level of Drp1, while increased Mfn2 in NRVMs after H/R injury, which indicates that TM may regulate mitochondrial dynamics during H/R injury of NRVMs.

Conclusions

TM exhibited cardiac protective effect on ischemic myocardium of rats after reperfusion and improved mitochondrial quality control through mitochondrial dynamics in NRVMs after H/R injury.

Abbreviations

I/R
ischemia/reperfusion
TM
Tongmai formula
NRVMs
neonatal rat ventricular myocytes
H/R
hypoxia/reoxygenation
JC-1
5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide
ROS
reactive oxygen species
TMRM
tetramethylrhodamine methyl ester
Mfn2
mitofusin 2
Drp1
dynamin related protein 1
mPTP
mitochondrial permeability transition pore
EF%
ejection fraction percentage
FS%
fraction shortening percentage
Peak Vel
peak velocity
dP/dt
maximum rate of left ventricular systolic pressure rises
TCC
2,3,5-triphenyltetrazolium chloride
H&E
hematoxylin and eosin
CK
Creatine kinase
LDH
lactate dehydrogenase
SOD
superoxide dismutase
MDA
malondialdyhyde
cTnT
cardiac troponin T
GSH
glutathione
DCFH-DA
2′,7’-dichlorofluorescin diacetate
TMRM
Tetramethylrhodamine methyl ester
ΔΨm
mitochondrial membrane potential
CYCS
cytochrome c
AIF
apoptosis inducing factor

Keywords

Mitochondrial quality control
Tongmai formula
Myocardial ischemia/reperfusion
Mitochondrial morphosis
Mitochondrial dynamics

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