Korean J Physiol Pharmacol.  2019 May;23(3):203-217. 10.4196/kjpp.2019.23.3.203.

Effects of heme oxygenase-1 upregulation on isoproterenol-induced myocardial infarction

• Eltobshy S
• Hussein AM
• Elmileegy
• Askar MH
• Khater Y
• Metias EF
• Helal GM

• KMID: 2443608
• DOI: http://doi.org/10.4196/kjpp.2019.23.3.203

Abstract

The present study was designed to examine the effect of heme oxygenase-1 (HO-1) induction by cobalt protoporphyrin (CoPP) on the cardiac functions and morphology, electrocardiogram (ECG) changes, myocardial antioxidants (superoxide dismutase [SOD] and glutathione [GSH]), and expression of heat shock protein (Hsp) 70 and connexin 43 (Cx-43) in myocardial muscles in isoproterenol (ISO) induced myocardial infarction (MI). Thirty two adult male Sprague Dawely rats were divided into 4 groups (each 8 rats): normal control (NC) group, ISO group: received ISO at dose of 150 mg/kg body weight intraperitoneally (i.p.) for 2 successive days; ISO + Trizma group: received (ISO) and Trizma (solvent of CoPP) at dose of 5 mg/kg i.p. injection 2 days before injection of ISO, with ISO at day 0 and at day 2 after ISO injections; and ISO + CoPP group: received ISO and CoPP at a dose of 5 mg/kg dissolved in Trizma i.p. injection as Trizma. We found that, administration of ISO caused significant increase in heart rate, corrected QT interval, ST segment, cardiac enzymes (lactate dehydrogenase, creatine kinase-muscle/brain), cardiac HO-1, Hsp70 with significant attenuation in myocardial GSH, SOD, and Cx-43. On the other hand, administration of CoPP caused significant improvement in ECG parameters, cardiac enzymes, cardiac morphology; antioxidants induced by ISO with significant increase in HO-1, Cx-43, and Hsp70 expression in myocardium. In conclusions, we concluded that induction of HO-1 by CoPP ameliorates ISO-induced myocardial injury, which might be due to up-regulation of Hsp70 and gap junction protein (Cx-43).

Keyword

Connexin 43; Heme oxygenase-1; HSP70 heat-shock proteins; Isoproterenol-myocardial infarction

MeSH Terms

Adult
Animals
Antioxidants
Body Weight
Cobalt
Connexin 43
Connexins
Creatine
Electrocardiography
Glutathione
Hand
Heart Rate
Heat-Shock Proteins
Heme Oxygenase-1*
Heme*
HSP70 Heat-Shock Proteins
Humans
Isoproterenol
Male
Muscles
Myocardial Infarction*
Myocardium
Oxidoreductases
Rats
Tromethamine
Up-Regulation*
Antioxidants
Cobalt
Connexin 43
Connexins
Creatine
Glutathione
HSP70 Heat-Shock Proteins
Heat-Shock Proteins
Heme
Heme Oxygenase-1
Isoproterenol
Oxidoreductases
Tromethamine

Figure

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  Fig. 1 Diagram of the study design. Times of drugs administration, electrocardiogram (ECG) recordings and collections of specimens is shown. Day −2, 2 days before isoproterenol (ISO) administration; day 0, day of first dose of ISO; day +1, day 1 after first dose of ISO; day +2, day 2 after ISO administration; day +5, day 5 after ISO administration. CoPP, cobalt protoporphyrin.

• 

  Fig. 2 Electrocardiogram records. From normal control (NC), isoproterenol (ISO), ISO + Trizma, and ISO + cobalt protoporphyrin (CoPP) groups at day 5.

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  Fig. 3 Effects of cobalt protoporphyrin (CoPP) on myocardial antioxidants. (A) Reduced glutathione (GSH) concentration (mg/g, heart tissue). (B) Superoxide dismutase (SOD) activity (U/g, heart tissue). *Significant vs. normal control (NC) group, #significant vs. isoproterenol (ISO) group, $significant vs. ISO + Trizma group at the same time interval.

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  Fig. 4 Relative expression of messenger RNA (mRNA) of heme oxygenase-1 (HO-1) in different groups. *Significant vs. normal control (NC) group, #significant vs. isoproterenol (ISO) group, $significant vs. ISO + Trizma group at the same time interval.

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  Fig. 5 Heart specimens (H&E staining, 400×). (A) Normal architecture of rat myocardium (normal control group). (B) Infracted zone with interstitial hemorrhage, necrotic changes (isoproterenol [ISO] group). (C) Marked neutrophil infiltration and extensive fibrillary degeneration and interstitial edema (ISO group). (D) Infracted zone with fibrillary degenerations and necrotic changes with extensive necrosis and interstitial edema and marked inflammatory infiltrates (ISO + Trizma group). (E) Mild interstitial edema with mild neutrophil infiltration (ISO + cobalt protoporphyrin [CoPP] group). (F) Normal architecture of myocardium (CoPP group).

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  Fig. 6 Expression of connexin 43 (Cx-43). (A) Graph showing the region of interest of Cx-43 expression in the myocardium from different groups. Immunohistochemical stained myocardial specimens (400×) showing a light brown color punctate distribution or rod-shaped pattern in end to end connection (intercalated disc) of Cx-43 (normal control group, B), less light brown color rod-shaped or punctate distribution pattern in end to end connection (intercalated disc) and side to side connection (isoproterenol [ISO] group, C), less light brown color rod-shaped or punctate distribution pattern in end to end connection (ISO + Trizma group, D), and side to side connection more light brown color punctate distribution or rod-shaped pattern in end to end connection (intercalated disc) and side to side connection (ISO + cobalt protoporphyrin [CoPP] group, E). *Significant vs. normal control group, #significant vs. ISO group, $significant vs. ISO + Trizma group at the same time interval.

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  Fig. 7 The expression of Hsp70 in heart tissues. (A) Region of interest ofheat shock protein 70 (Hsp70) expression in myocardium in different groups. Immunohistochemical stained myocardial fibers sections for Hsp70 (400×) showing light brown color in cardiomyocytes cytoplasm (normal control group, B), moderate brown color in cardiomyocytes cytoplasm (isoproterenol [ISO] group, C), moderate brown color in cardiomyocytes cytoplasm (ISO + Trizma group, D), and marked brown color in cardiomyocytes cytoplasm (ISO + cobalt protoporphyrin group, E). *Significant vs. normal control group, #significant vs. ISO group, $significant vs. ISO + Trizma group at the same time interval.

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  Fig. 8 Graphical abstract showing the proposed mechanisms of isoproterenol (ISO)-induced myocardial infarction (MI) and role of heme oxygenase-1 (HO-1) upregulation. ISO stimulates β1 adrenergic receptors (β1-ARs) which stimulates adenylate cyclase and inhibits phosphodiesterase activity leads to increased cyclic adenosine monophosphate (cAMP) level which result in enhance Ca+2 inflow (via stimulation of L-type Ca+2 channels) results in Ca+2 overload and stimulates p38 mitogen activated protein kinase (MAPK) activity. A raised cytoso1ic Ca+2 may cause myocardial cell death by excessive activation of Ca+2-dependent ATPases and impairing mitochondrial phosphorylation and generation of reactive oxygen species (ROS). Also ISO produces oxidative stress via formation of adrenochromes (quinones) through its autooxidation. ROS causes lipid peroxidation and cell damage and dephosphorylation of gap junctions connexin-43 (Cx-43) leading to cardiac arrhythmias. Also ROS leads to unfolding and aggregation of protein. However, ROS causes nuclear factor erythroid 2-related factor 2 (Nrf2) dissociates from kelch-like ECH-associated protein-1 (Keap-1) and translocates to the nucleus, where binding of Nrf2 to the antioxidant response elements promoter site, leads to the upregulation of HO-1 gene expression. Also cobalt protoporphyrin (CoPP) stimulates upregulation of Nrf2 protein, which induces HO-1 gene expression. HO-1 decreases ROS by its antioxidant action preserving Cx-43 architecture and stimulates HSF1 releasing from the complex with heat shock protein (Hsp), and then it enters the nucleus to bind heat shock elements in the promoter region of HSP70 genes, which leads to their transcription. HSP70 act as molecular chaperons converting aggregates to folded protein.

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