Despite the significant advances that have been achieved in the management of coronary heart disease (CHD), it remains the leading cause of mortality and morbidity worldwide [1]. The main cause of death from CHD is myocardial infarction (MI), which is an ischemic necrosis of cardiac myocytes results from prolonged interruption of blood supply to certain area of heart [2]. Isoproterenol (ISO)-induced MI is a well-established animal model of pharmacologically induced MI [3]. Several hypotheses have been postulated to explain pathophysiology of the myocardial injury in ISO-induced MI such as relative myocardial hypoxia due to excessive cardiac work and myocardial O₂ demands which is intensified by hypotension or hypoxia due to direct O₂-wasting effect of catecholamines or their autoxidation products [4].

Heme oxygenase (HO), a key-regulating enzyme in heme degradation, catalyzes the oxidation of heme to produce several active molecules such as ferrous iron (Fe⁺²), biliverdin and carbon monoxide (CO) [5]. Heme oxygenase-1 (HO-1) is normally expressed at low levels in most of organs; however, it is highly inducible in response to a variety of stimuli to protect cells against oxidative and inflammatory injury [6]. It has several cytoprotective properties such as anti-oxidative, anti-inflammatory and antiapoptotic effects [6]. It has been found that over-expression of HO-1 in animals in either transgenic mouse [7] or transfected rat [8] or by pre-treatment with cobalt protoporphyrin IX (CoPP) [9], a powerful inducer of HO-1 [10], reduced infarct size, ventricular remodeling, enhanced endothelial function, promoted neoangiogenesis and restored cardiac metabolism [11]. However, the role of HO-1 upregulation in modulation of the electrical activities of the cardiac muscles and in induction of molecular cytoprotective chaperones (heat shock proteins, Hsps) in MI needs more clarification.

The intercellular-junctions are group of proteins such as connexin 43 (Cx-43) that electrically couple cardiomyocytes for its coordinated depolarization, and its uncoupling has been associated with heart diseases [12]. Also, Hsps are endogenous proteins that act as intracellular chaperones for other proteins through its essential role in protein corrective function (help protein folding and correct its shape), and preventing the unwanted protein aggregations due to the process of aging and in settings of cardiac stress [13]. The most important and best studied class of all Hsps is the 70 kDa (kilodalton) family [13]. We postulated that the induction of HO-1 could improve electrical activity of the cardiac muscle through up regulation of Cx-43 and Hsp70 in MI. So, the aim of the present study was to investigate the effects of HO-1 induction on the cardiac functions and morphology, electrocardiogram (ECG) changes, oxidative stress markers, and expression of Cx-43 and Hsp70.

The main findings of the present study include i) administration of ISO at a dose of 150 mg/kg body weight caused significant deteriorations in cardiac morphology, ECG parameters and cardiac enzymes with reduction of the endogenous antioxidants and Cx-43 and mild upregulation of HO-1 and Hsp70 in heart tissues; and ii) upregulation of HO-1 by CoPP was associated significant improvement in cardiac enzymes, cardiac morphology, disturbed ECG parameters, endogenous antioxidants and expression of Cx-43 and Hsp70 in myocardium. We hypothesized that HO-1 induction by CoPP could improve the cardiac functions, morphology and ECG changes in ISO-induced MI.

In the present study, we found mild upregulation of HO-1 in heart tissues in ISO group. This effect might be due to oxidative stress induced by large dose of ISO or due to direct effect of ISO. In line with this hypothesis, Hu et al. [19] and Otterbein et al. [20] demonstrated that ISO could be induced in response to a variety of stimuli and injury, such as oxidative stress and myocardial ischemia/reperfusion (I/R) injury in several organs and cell types. Also, activation of β1-adrenoreceptors by ISO causes significant increase in cyclic adenosine monophosphate (cAMP), which in turn activates protein kinase A [21], and stimulates phosphoinositide 3-kinase (PI3K) and p38 mitogen activated protein kinase (MAPK) activity. Activated PI3K and p38MAPK make the transcription factor (nuclear factor-erythroid-related factor 2, Nrf2) to move to nucleus, where binding of Nrf2 to the antioxidant response elements promoter site, leads to the upregulation of HO-1 gene expression [22]. Moreover, the present study confirmed upregulation of HO-1 by CoPP administration. We showed that administration of CoPP at a dose of 5 mg/kg body weight every 48 h caused significant upregulation of HO-1 at the level of mRNA. The dose of 5 mg/kg and regimen of CoPP administration, i.e. single dose every 48 h were based on previous studies which reported that the maximum upregulation of HO-1 occurs within 48 h after CoPP administration [1516]. Previous studies demonstrated similar findings of HO-1 upregulation by metalloprotophyrins (CoPP) in other tissues such as kidney [14,23]. The upregulation of HO-1 by ISO was mild compared to that induced by CoPP as well as it was associated with marked increase in oxidative stress, that is why it had no protective effect.

In ECG recording, the present study found that ISO administration caused significant increase of the HR, prolonged cQT and elevated ST segment which are diagnostic features for MI [24]. These results are in line with those reported by Thippeswamy et al. [25] and Metias et al. [17]. Tachycardia demonstrated in ISO group could be due to disturbed sympathetic and parasympathetic inputs to the heart due to excessive stimulation of β1-adrenorceptor by ISO which also, causes deterioration of mitochondrial energetic and suppression of Ca⁺² transport which causes intracellular Ca⁺² overload [26]. Also, prolongation of QT interval in ISO group in the present study may be related to cardiac vagal dysfunction [27] or reduction of gap junction protein Cx-43 expression and their disorganization as shown in the present study. Also, ST-segment showed an elevation in ISO group which reflects myocardial inflammation and damage which causes “injury currents” from damaged cells that are partially depolarized to the healthy myocardium [28]. Moreover, we found in the present study, administration of CoPP significantly attenuated ISO-induced ECG changes suggesting that CoPP administration and HO-1 upregulation improved the myocardial electrical activity in ISO-induced MI. This is confirmed by the negative correlation between HO-1 and HR, ST segment elevation and duration of cQT interval. In line with these findings, Kusmic et al. [11], Pósa et al. [29] and Ndisang [30] demonstrated that HO-1 induction reduced the size and the thickness of the myocardial infarct, as well as preserved Cx-43 architecture in the remote zone.

The next point in the present study was to investigate the effects of ISO on serum levels of CK-MB and LDH. CK-MB isoenzyme activity is useful as an index for the early diagnosis of MI because it is present mainly in myocardial tissues [31]. Also, serum CK-MB is considerably more specific for myocardial damage than the serum total CK [32]. It has reported that serum CK-MB concentration rise starts to develop 4 ± 6 h after injury, reaching its peak after 12 ± 24 h and decrease after 48 ± 72 h. The present study demonstrated that significant increase serum CK-MB at 24 h after second dose of ISO, i.e. at day 2 with significant reduction at day 5. Similar findings were reported by Bodor [33] and Metias et al. [17]. The significant elevation in CK-MB could be due to leakage of CK-MB enzymes via damaged myocardial cell membrane or its rupture due to necrotic cell death. The same changes occur in serum LDH which is a cytosolic enzyme that is present in all the tissues involved in glycolysis. LDH activity increases after about 18 h post MI, peaks between the second and third day and remains elevated for about a week [30]. Our results are in line with those reported by Bodor [33] and Metias et al. [17]. Panda and Naik [34] explained the significant elevation in serum LDH in ISO group by necrotic damage of myocardial cells by reactive oxygen species (ROS) as shown in our study. In line with biochemical changes in ISO group, histopathological examination revealed significant myocardial damage in the form of focal necrotic lesions, interstitial hemorrhage and edema with marked inflammatory cell infiltrates (polymorph granular leucocytes and neutrophils). These findings are in agreement with those reported by Zhou et al. [35], Liu et al. [36] and Metias et al. [17]. Myocardial damage and necrosis could be explained by excess O₂ consumption, poor O₂ utilization, cytosolic overload and accumulation of Ca²⁺, excessive production of ROS, altered membrane permeability, altered myocardial cell metabolism, increased myocardial cAMP levels and intracellular acidosis [313738].

On the other hand, we found that administration of CoPP caused significant improvement in the serum levels of cardiac enzymes (LDH, CK-MB) and cardiac morphology suggesting cardioprotective effects for CoPP and HO-1 upregulation against ISO-induced MI. Moreover, the negative correlation between serum LDH and CK-MB levels and HO-1 level might confirm this suggestion. In line with these findings, Woo et al. [39] demonstrated that overexpression of HO-1 was associated with longterm acceptance of mouse-to-rat cardiac transplants. Also, Katori et al. [40] demonstrated that donor hearts from HO-1-deficient donors were vigorously rejected. In kidney tissues, Barakat et al. [14] demonstrated upregulation of HO-1 by CoPP had renoprotective effect against renal ischemia. Also, chronic induction of HO-1 showed hepatoprotective effects against liver ischemia in rats [41] and controlled the renal hemodynamics and excretory functions during renal ischemia [42].

Myocardial oxidative stress in ISO-induced MI results from disturbed physiological balance between formation of free radicals and anti-oxidative defense system [35] via formation of adrenochromes such as quinones through auto-oxidation of ISO which are implicated for generation of highly toxic O₂ free radicals such as superoxide (O₂−) and hydrogen peroxide (H₂O₂) [43]. Also, Ca²⁺ overload and their consequences are crucial in ISO-induced injury and generation of ROS [44]. ROS attack the cell membrane phospholipids and fatty acids producing lipid peroxides that are longer-lived species than ROS and can initiate the chain reactions that enhance the oxidative damage to the cells [45]. Moreover, this oxidative stress causes depletion of the anti-oxidant systems such as reduced GSH and SOD as shown in the present study. The reduction in GSH levels observed in ISO-induced MI was either due to excessive destruction or decreased GSH synthesis. The concentration of GSH is kept constant inside the cells by the activity of glutathione peroxidase enzyme which convert the oxidized form of glutathione to reduced form (GSH) [34]. Also, reduction of the activity of the SOD in ISO-induced MI might be due to the increased generation of reactive oxygen radicals, such as O₂⁻ and H₂O₂, which in turn leads to the inactivation of this enzyme activity.

HO-1 plays an important role in maintaining redox status within the heart, thereby protecting against oxidative insult. In the present study demonstrated that CoPP treatment caused significant increase in antioxidant (GSH and SOD) in the myocardium than ISO group. Moreover, there are positive correlation between HO-1 and GSH and SOD activity. These findings suggest antioxidant role for CoPP administration. In consistence with these findings, Barakat et al. [14] demonstrated significant reduction in malondialdehyde (marker of lipid peroxidations) and increase in antioxidants such as SOD in kidney tissues during renal I/R injury. This effect may be due to HO-1 is a major antioxidant enzyme that plays a vital role in the defense against free radical-induced oxidative damage in human skin cells [46]. Moreover, Shan et al. [47] reported that COPP induces up-regulation of the transcription factor nrf-2 which in turn induces many antioxidant defense mechanisms to mitigate oxidative damage and maintain the cellular redox homeostasis [48].

Gap junction proteins such as Cx-43 are predominately expressed in the intercalated discs and to less extent at the lateral sides of the ventricular myocytes [49]. It has been demonstrated that myocardial ischemia causes redistribution of Cx-43 along the lateral plasma membrane away from the intercalated disk region [50]. In the present study we reported similar findings in ISO-induced myocardial injury. The lateralized Cx-43 is dephosphorylated, whereas phosphorylated forms remain at the intercalated disk region [51]. These ischemic changes in Cx-43 phosphorylation are followed by loss of Cx-43 due to degradation [52] leading to development of cardiac arrhythmias. In the present study we demonstrated significant reduction in Cx-43 expression in ISO group which partially explain the cardiac arrhythmic changes induced by ISO. Metias et al. [17] reported similar changes in Cx-43 in ISO-induced MI. Also, Sun et al. [53] concluded that the reduction and redistribution of myocardial Cx-43 might be mediated by ATP-sensitive potassium channels through preserving protein kinase C-dependent connexion-43 level after MI. Disruption of myocardial Cx-43 in the present study might be explained by enhanced oxidative stress in the heart tissues. In our study we demonstrated upregulation of Cx-43 in CoPP-treated rats as well as we found positive correlation between HO-1 and Cx-43 expression suggesting that upregulation of gap junction protein Cx-43 might represent a possible underlying mechanism for the cardioprotective role of CoPP administration and HO-1 upregulation. In agreement with these findings, Lakkisto et al. [15] demonstrated that Wistar rats treated with hemin (a potent inducer of HO-1) prevented the arrhythmias and the reduction in total Cx-43 induced by I/R injury. Also, Schulz et al. [54] found that hypoxia cause dephosphorylation of Cx-43, whereas ischemic preconditioning with the HO-1 inducer hemin preserves phosphorylation of Cx-43 and reduces infarct size in I/R pig hearts.

Shock proteins (Hsps) are intracellular cytoprotective proteins which are essential for cell survival both in physiological and stress conditions [55]. The most important cytoprotective Hsp is Hsp70. It has been reported that overexpression of Hsp70 could protects the heart against damaging effects of ischemia. Also, Hsp70 overexpression represents a protective mechanism not only in acute settings, e.g. I/R injury after coronary bypass grafting, but also in patients with chronic “stress”, as in congestive heart failure [56]. So, overexpression of Hsp70 could represent an innate protective mechanism which helped to restore physiological conditions [55]. In line with these findings, we reported there was upregulation of Hsp70 in myocardium compared to normal hearts. These findings are parallel with those reported by Li et al. [57]. In agreement with our results, Tanwar et al. [58] demonstrated that ISO and norepinephrine can induce production of Hsp70 in angiotensin II-induced myocardial injury via activation of sympatho-adrenal system. Also, Tanonaka et al. [59] reported that reduction of Hsp70 in heart tissues may play a significant role in reduction of the contractile function during the development of heart failure in a rat model. We postulated that the upregulation of Hsp70 in the present study was by increased oxidative stress in myocardium. Also, in the present study, we demonstrated that CoPP treatment caused significant increase in Hsp70 in heart tissues and positive correlation between HO-1 and Hsp70 suggesting that upregulation of Hsp70 might be a potential mechanism for the cardioprotective action of CoPP and HO-1 induction in ISO-induced MI. In fact, HO-1 was previously known as Hsp32 which is a member of Hsp family.

Another mechanism for HO-1 upregulation explained its cytoprotective such as its antiapoptotic action. Lakkisto et al. [60] demonstrated that upregulation of HO-1 inhibits apoptosis both in vitro and in vivo. Also, Katori et al. [40] found that CoPP administration decreased frequency of apoptotic cells (both myocyte and endothelial) in cardiac isografts. Indeed, adjunctive treatment with zinc protoporphyrin, an HO-1 enzyme activity inhibitor, prevented the expression of B-cell lymphoma 2 (Bcl-2) and delayed the expression of Bag-1 (Bcl-2 associated athanogene 1) antiapoptotic [40]. However, this mechanism was not investigated in the present study which may be considered as a limitation of this study and further studies may be needed to explore this mechanism for HO-1 upregulation.

We concluded that administration of ISO at a dose of 150 mg/kg caused marked deteriorations in cardiac functions and morphology, which was associated with signs of MI in ECG, reduction of myocardial antioxidants, Cx-43 proteins and upregulation of Hsp70. Upregulation of HO-1 by CoPP had a cardioprotective action against ISO induced myocardial injury, which might be due to increased myocardial antioxidant capacity and upregulation of gap junction proteins (Cx-43) and Hsp70 (see Fig. 8, graphical abstract).