Ischemic heart disease is responsible for more than one-third of worldwide mortality [1, 5]. MI has been ranked as one of the most important ischemic pathologies which causes extensive cardiomyocyte death, leading to LV remodeling and HF [2, 4, 26]. As a result, comprehensive studies of potential molecular signaling and associated mechanisms during post-MI progression would be beneficial in informing the development of new therapeutic strategies and therapies in the clinical setting of post-MI patients. In this study, we performed permanent LAD ligation to mimic MI pathology in rats. The main outcomes are as follows: (i) Post-MI mediated myocardial injury, cardiac systolic and diastolic dysfunction, and subsequently HF; (ii) Post-MI impaired cardiac mitochondrial dynamic balance by increasing mitochondrial fission but decreasing mitochondrial fusion, which in turn impaired mitochondrial function; (iii) Post-MI mediated several PCDs, including apoptosis, necroptosis, and pyroptosis, but not ferroptosis; (iv) pyroptosis was the most dominant form of PCD in post-MI pathology.
HF is acknowledged as being the fatal consequence in about half of all patients with an unfavorable prognosis of MI [27, 28]. HF could develop soon after acute MI or gradually post-MI . In the current study, our results confirmed that post-MI there were adverse biomechanical changes in the heart, resulting in impaired cardiac contractile function in both systolic and diastolic phases. Similarly, post-MI-induced cardiac geometry changed by the induction of LV structural thinning and dilatation. As a consequence of sustained ischemic insults, myocardial injury and cardiomyocyte death occurred, as indicated by elevated levels of NT-proBNP and cTnI in peripheral blood, which were associated with LV wall stress and myocardial cell injury. An increase in myocardial injury post-MI has also been reported to be associated with an increase in oxidative stress, inflammation, and mitochondrial dysfunction, resulting in cell death in the ischemic myocardium [30, 31].
Mitochondrial function is governed by a morphologically controlled mechanism of mitochondria, known as ‘mitochondrial dynamics’ in which fission and fusion processes are coordinated [17, 32]. These phenomena ensure the appropriate balance of energy demand and supply in the mitochondria. Previous studies have reported that excessive mitochondrial fission and decreased mitochondrial fusion increased the severity of HF [15,16,17,18,19]. Consistent with these studies, we observed a significant increase in the fission markers (pDRP1/tDRP1 ratio) but a decrease in the fusion (MFN1 and OPA1) markers in post-MI rats, indicating a dynamic imbalance of mitochondria after MI. This finding was consistent with a previous report which demonstrated that OPA1 required MFN1, but not MFN2, to regulate the fusion process .
To date, the manifestations and mechanisms of various PCDs, including apoptosis, necroptosis, ferroptosis, and pyroptosis, have been identified as being associated with cardiovascular diseases [6,7,8,9, 11, 12]. Previous studies have shown that apoptosis was upregulated in the first 2 h and persisted up to 12 weeks after MI [9, 11, 26]. Wang X. and colleagues (2018) found that upregulation of apoptotic signaling, including Cleaved-caspase 3 and TUNEL positive cardiomyocytes, was detected 2-weeks after MI and remained elevated for weeks in the mouse model of post-MI . In our study, we found a significant increase in apoptosis-related proteins, including Cytochrome C, Bax, and Cleaved-caspase3 in rats 5-weeks after MI. It is well known that apoptosis is responsible for early cardiomyocyte death and would trigger macrophage clearance but is less likely to induce inflammation . In contrast to apoptosis, necroptosis is considered as a potent inflammatory inducer . It has been shown that myocardial necroptosis was detected 1 to 12 weeks after MI in the mouse model of post-MI, as indicated by increased tRIPK1, tRIPK3, and tMLKL . Consistently, we observed a marked increase in pRIPK3 and pMLKL protein levels, suggesting activation of necroptosis.
Ferroptosis is a novel iron-dependent form of PCD characterized by the accumulation of ROS, leading to lipid peroxidation of polyunsaturated fatty acids in plasma membranes, resulting in membrane breakdown [6, 7, 35]. Glutathione peroxidase 4 (GPX4) serves as an endogenous regulator of lipid peroxidation. The depletion of GPX4 would lead to lipid peroxidation and ferroptosis [7, 35]. It has been reported that acute oxidative injury exacerbated myocardial ferroptosis in SD rats after 1-week MI . In our study, the MI rats had significantly higher MDA levels in both cardiac tissue and serum than sham rats, which indicated lipid peroxidation. Surprisingly, we also found that the expression of GPX4 levels were increased in MI rats, whereas no significant change in the expression of ACSL4 was found. These findings suggested that ferroptosis was declining in 5-weeks MI. This is confirmed by a previous report demonstrating that GPX4 levels were decreased in the acute and middle stages of MI (i.e., 1 day to 1 week after LAD ligation), but subsequently increased in the late phase (i.e. 8 weeks of MI) . Taken together, all of these findings suggested that ferroptosis was declining due to the compensatory increased antioxidant regulation of GPX4 after MI.
Pyroptosis is known as an inflammation mediated cell death as a result of the priming and activating of the inflammasome, which subsequently increases the permeability of the plasma membrane and releases inflammatory cytokines . Inflammasome activation and pyroptotic cell death have been identified in various cardiovascular disease models, including acute MI, cardiac I/R, and HF [8, 37,38,39]. Prior to this report, the current understanding of the molecular phenotype of the dominant PCDs involved in post-MI was still elusive.
Our findings revealed for the first time that the pyroptosis executor protein GSDMD-NT was also upregulated in the post-MI model. In summary, we demonstrated that chronic progression of MI mediates multiple PCDs, including apoptosis, necroptosis, and pyroptosis, but not ferroptosis. And pyroptosis was the most dominant form of PCD in post-MI pathology.
This study provided the first insights on a significant escalation of pyroptosis activation as a candidate therapeutic target for post-MI, together with the demonstration of the mechanistic insights across different modes of programmed cell death and mitochondrial involvement. Our findings highlight a potential future therapeutic opportunity for pyroptosis, which could represent a novel therapeutic approach for post-MI patients. However, our preliminary findings used a small sample size. Also, the potential benefits of specific inhibition of each PCDs in post-MI pathology were not investigated in this study. Hence, future studies using various inhibitors of PCDs are needed to warrant its translation to a clinical setting of post-MI.