Quantification of Murine Myocardial Infarct Size using 2D and 4D High Frequency Ultrasound

Background: Ischemic heart disease is the leading cause of death in the United States, Canada, and worldwide. Severe disease is characterized by coronary artery occlusion, loss of blood flow to the myocardium, and necrosis of tissue, with subsequent remodeling of the heart wall, including fibrotic scarring. The current study aims to demonstrate the efficacy of quantitating infarct size via 2D echocardiographic akinetic length and 4D echocardiographic infarct volume and surface area as in vivo analysis techniques. We further describe and evaluate a new surface area strain analysis technique for estimating myocardial infarction (MI) size after ischemic injury. Methods: Experimental MI was induced in mice via left coronary artery ligation. Ejection fraction and infarct size were measured through 2D and 4D echocardiography. Infarct size established via histology was compared to ultrasound-based metrics via linear regression analysis. Results: 2D echocardiographic akinetic length (r = 0.76, p = 0.03), 4D echocardiographic infarct volume (r = 0.85, p = 0.008) and surface area (r = 0.90, p = 0.002) correlate well with histology. While both 2D and 4D echocardiography were reliable measurement techniques to assess infarct, 4D analysis is superior in assessing asymmetry of the left ventricle and the infarct. Strain analysis performed on 4D data also provides additional infarct sizing techniques, which correlate with histology (surface strain: r = 0.94, p < 0.001, transmural thickness: r = 0.76, p = 0.001). Conclusions: 2D echocardiographic akinetic length, 4D echocardiography ultrasound and strain provide effective in vivo methods for measuring fibrotic scarring after MI.

Evaluation of Cardiovascular Experiment Animal

In vivo models of myocardial infarction induced by coronary artery ligation in rats usually suffer from high early mortality and a low rate of induction. This study investigated the time course initiation of chronic myocardial infarction in albino rats and the possibility of reducing early mortality rate due to myocardial infarction by modification of the surgical technique. CAL was carried out by passing the suture through the pericardial layer around the midway of the left anterior descending coronary artery including a small area of the myocardium to avoid mechanical damage to the heart geometry. In addition, the role of endothelin-1 in rat heart with congestive heart failure was critically assessed. Time course initiation experiments were designed by sacrificing the animals at different time intervals and by carrying out physiological, biochemical, histopathological, electron microscopical and immunohistochemical studies. Specific markers of myocardial injury, viz. cardiac troponin-T, high sensitivity C-reactive protein, lactate dehydrogenase and fibrinogen were measured at different time points. Serum marker enzymes and activities of lysosomal hydrolases were found to be elevated on the eighth day post-ligation. Histopathological studies demonstrated focal areas showing fibrovascular tissue containing fibroblasts, collagenous ground substance and numerous small capillaries replacing cardiac muscle fibers. Transmission electron micrographs exhibited mitochondrial changes of well-developed irreversible cardiac injury, viz. swelling, disorganization of cristae, appearance of mitochondrial amorphous matrix densities, and significant distortion of muscle fibers and distinct disruption of the intercalated discs. Immune blotting studies confirmed the presence of alpha 2-macroglobulin which supported the inflammatory response. The severity of the CMI was inferred by the measurement of the level of ET-1 in plasma and left ventricle which was significantly higher in the CMI rats than in the sham-operated rats. Immunohistochemical studies at different time intervals showed that there was a significant immunoexpression of ET-1 on the eighth day post-ligation. This study conclusively showed that ligation of left anterior descending artery minimised mortality and ET-1 was expressed during CMI.

Attenuation of Adverse Postinfarction Left Ventricular Remodeling with Empagliflozin Enhances Mitochondria-Linked Cellular Energetics and Mitochondrial Biogenesis

Sodium–glucose cotransporter 2 (SGLT2) inhibitors such as empagliflozin are known to reduce the risk of hospitalizations related to heart failure irrespective of diabetic state. Meanwhile, adverse cardiac remodeling remains the leading cause of heart failure and death in the USA. Thus, understanding the mechanisms that are responsible for the beneficial effects of SGLT2 inhibitors is of the utmost relevance and importance. Our previous work illustrated a connection between adverse cardiac remodeling and the regulation of mitochondrial turnover and cellular energetics using a short-acting glucagon-like peptide-1 receptor agonist (GLP1Ra). Here, we sought to determine if the mechanism of the SGLT2 inhibitor empagliflozin (EMPA) in ameliorating adverse remodeling was similar and/or to identify what differences exist, if any. To this end, we administered permanent coronary artery ligation to induce adverse remodeling in wild-type and Parkin knockout mice and examined the progression of adverse cardiac remodeling with or without EMPA treatment over time. Like GLP1Ra, we found that EMPA affords a robust attenuation of PCAL-induced adverse remodeling. Interestingly, unlike the GLP1Ra, EMPA does not require Parkin to improve/maintain mitochondria-related cellular energetics and afford its benefits against developing adverse remodeling. These findings suggests that further investigation of EMPA is warranted as a potential path for developing therapy against adverse cardiac remodeling for patients that may have Parkin and/or mitophagy-related deficiencies.

Sympathetic Nerve Innervation and Metabolism in Ischemic Myocardium in Response To Remote Ischemic Conditioning

Background Multiple potential interventions have been tested to protect the heart against myocardial ischemia/reperfusion (MIR) injury. Remote ischemic conditioning (RIC), an endogenous cardioprotective approach, could markedly improve cardiac function post-myocardial ischemia injury. In this study, we aimed to assess the effects of RIC on cardiac sympathetic nerve innervation and metabolism in the association with Chondroitin sulfate proteoglycans (GSPG). Methods Transient myocardial ischemia (30 min) is induced by ligature of the left anterior descending coronary artery ligation (LAD) in male Sprague Dawley rats (250-350 g), in vivo cardiac [11C]mHED and 2-[18F]FDG PET scans were performed at 14 days after ischemia. Remote ischemic preconditioning (RIPerc) was induced by three cycles of five-minute-long unilateral hind limb ischemia and intermittent five minutes of reperfusion during LAD occlusion period. The quantitative parameters were quantified in parametric polar maps. This standardized format facilitates the regional radioactive quantification of parameters in deficit regions to remote areas. The ex vivo radionuclide distribution was additionally identified using autoradiography. Myocardial neuron density and GSPG expression were assessed by immunohistochemistry. Results There was no significant difference in the metabolism-defected to the remote activity ratio (44.6±4.8% vs. 45.4±4.4%) between control rats (MIR) and treated (MIR+RIPerc) rats (P>0.05). Additionally, the mean nervous activity of denervated myocardium activity was significantly elevated in rats with RIPerc coupled with reduced denervated myocardium size compared to the rats MIR group (35.9±7.1% vs. 28.9±2.3% of the left ventricular (LV) remote area (P<0.05). These findings were associated with preserved LV systolic function and a significant reduction in GSPG expression in the myocardium. Conclusion RIPerc presented the effect on cardiac sympathetic nerve innervation following ischemia, but there is no significant effect on myocardial metabolism. A long-term outcome study is warranted.

Differential Regulation of Myocardial E3 Ligases and Deubiquitinases in Ischemic Heart Failure

The pathological changes of ubiquitination and deubiquitination following myocardial infarction (MI) and chronic heart failure (CHF) have been sparsely examined. We investigated the expression of muscle-specific E3 ubiquitin ligases and deubiquitinases in MI and CHF. Therefore, mice were assigned to coronary artery ligation for 3 days or 10 weeks as well as for sham operation (each n = 10). Expression of E3 ligases (MAFBX, MURF1, CHIP, ITCH, MDM2) and deubiquitinases (A20, CYLD, UCH-L1, USP14, USP19) was determined. After MI and in CHF, the mRNA expression of MURF1, CHIP and MDM2 (all p < 0.05) was decreased. Protein expression analyses revealed that ITCH expression decreased in CHF (p = 0.01), whereas MDM2 expression increased in MI (p = 0.02) and decreased in CHF (p = 0.02). Except for USP19 mRNA expression that decreased at 3 days and 10 weeks (both p < 0.01), the expression of other deubiquitinases remained unaffected after MI and CHF. The expression of myocardial E3 ligases is differentially regulated following MI, raising the question of whether an upstream regulation exists that is activated by MI for tissue protection or whether the downregulation of E3 ligases enables myocardial hypertrophy following MI.

Proteomic Sequencing of Stellate Ganglions in Rabbits With Myocardial Infarction

The stellate ganglion (SG) of the autonomic nervous system plays important role in cardiovascular diseases (CDs). Myocardial infarction (MI) is associated with sustained increasing cardiac sympathetic nerve activity. Expressions and functions of proteins in SG tissue after MI are remaining unclear. This study is to explore the expression characteristics of proteins in SGs associated with MI. Japanese big-ear white rabbits (n = 22) were randomly assigned to the control group and MI group. The MI model was established by left anterior descending coronary artery ligation and confirmed by serum myocardial enzymes increasing 2,3,5-triphenyltetrazolium (TTC) staining and echocardiography. The expressions of proteins in rabbit SGs after MI were detected using tandem mass tags (TMT) quantitative proteomic sequencing. There were 3,043 credible proteins were predicted in rabbit SG tissues and 383 differentially expressed proteins (DEPs) including 143 upregulated and 240 downregulated proteins. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DEPs involved in adrenergic signaling in cardiomyocytes, positive regulation of ERK1 and ERK2 cascade, and other biological processes. Three kinds of proteins directly correlated to CDs were selected to be validated by the subsequent western blot experiment. This study first identified the characterization of proteins in rabbit SG after MI, which laid a solid foundation for revealing the mechanism of roles of SG on the MI process.

Enhanced Expression and Function of Renal SGLT2 (Sodium-Glucose Cotransporter 2) in Heart Failure: Role of Renal Nerves

Background: Recent clinical studies demonstrate that SGLT2 (sodium-glucose cotransporter 2) inhibitors ameliorate heart failure (HF). The present study was conducted to assess the expression and function of renal SGLT2 and the influence of enhanced renal sympathetic tone in HF. Methods: Four weeks after coronary artery ligation surgery to induce HF, surgical bilateral renal denervation (RDN) was performed in rats. Four groups of rats (Sham-operated control [Sham], Sham+RDN, HF and HF+RDN; n=6/group) were used. Immunohistochemistry and Western blot analysis were performed to evaluate the renal SGLT2 expression. One week after RDN (5 weeks after induction of HF), intravenous injection of SGLT2 inhibitor dapagliflozin were performed to assess renal excretory responses. In vitro, human embryonic kidney cells were used to investigate the fractionation of SGLT2 after norepinephrine treatment. Results: In rats with HF, (1) SGLT2 expression in the proximal tubule of the kidney was increased; (2) the response of increases in urine flow, sodium excretion, and glucose excretion to dapagliflozin were greater; and (3) RDN attenuated renal SGLT2 expression and normalized renal functional responses to dapagliflozin. In vitro, norepinephrine promoted translocation of SGLT2 to the cell membrane. Conclusions: These results indicate that the enhanced tonic renal sympathetic nerve activation in HF increases the expression and functional activity of renal SGLT2. Potentiated trafficking of SGLT2 to cell surface in renal proximal tubules mediated by norepinephrine may contribute to this functional activation of SGLT2 in HF. These findings provide critical insight into the underlying mechanisms for the beneficial effects of SGLT2 inhibitors on HF reported in the clinical studies.

It takes time to heal a broken heart: Ventricular plasticity improves heart performance after myocardial infarction in rainbow trout, Oncorhynchus mykiss

Coronary arteriosclerosis is a common feature of both wild and farmed salmonid fishes and may be linked to stress-induced cardiac pathologies. Yet, the plasticity and capacity for long-term myocardial restructuring and recovery following a restriction in coronary blood supply is unknown. Here, we analyzed the consequences of acute (3 days) and chronic (from 33 to 62 days) coronary occlusion (i.e., coronary artery ligation) on cardiac morphological characteristics and in vivo function in juvenile rainbow trout, Oncorhynchus mykiss. Acute coronary occlusion resulted in elevated resting heart rate and decreased inter-beat variability, which are both markers of autonomic dysfunction following acute myocardial ischemia, along with severely reduced heart rate scope (maximum – resting heart rate) relative to sham-operated trout. We also observed a loss of myocardial interstitial collagen and compact myocardium. Following long-term coronary- ligation, resting heart rate and heart rate scope normalized relative to sham-operated trout. Moreover, a distinct fibrous collagen layer separating the compact myocardium into two layers had formed. This may contribute to maintain ventricular integrity across the cardiac cycle, or alternatively, demark a region of the compact myocardium that continues to receive oxygen from the luminal venous blood. Taken together, we demonstrate that rainbow trout may cope with the aversive effects caused by coronary artery obstruction through plastic ventricular remodeling, which, at least in part, restores cardiac performance and myocardium oxygenation.

LRRK2 Deficiency Protects Hearts from Myocardial Infarction Injury in Mice via the P53/HMGB1 Pathway

Purpose: LRRK2 is a Ser/Thr kinase with multiple functional domains. Current studies have shown that its mutations are closely related to hereditary Parkinson's disease. However, its role in cardiovascular disease, especially in myocardial infarction, is unclear. The aim of this study was to explore the functional role of LRRK2 in myocardial infarction. Methods: Wild-type and LRRK2 knockout mice were subjected to coronary artery ligation (left anterior descent) to establish a myocardial infarction mouse model. Neonatal rat cardiomyocytes were subjected to hypoxia to induce hypoxia injury in vitro. Results: We found increased LRRK2 expression levels in the infarct periphery of mouse hearts and hypoxic cardiomyocytes. LRRK2-deficient mice exhibited a decreased death rate and reduced infarction area compared to the wild-type controls 14 days after infarction. LRRK2-deficient mice showed reduced left ventricular fibrosis and inflammatory response, as well as improved cardiac function. In the in vitro study, LRRK2 silencing decreased the cleaved-caspase3 activity, reduced cardiomyocyte apoptosis, and diminished hypoxia-induced inflammation. However, LRRK2 overexpression enhanced the cleaved-caspase3 activity, increased the number of apoptotic cardiomyocytes, and caused remarkable hypoxia-induced inflammation. When exploring the related underlying mechanisms, we found that hypoxia induced an increase in HIFα expression, which enhanced LRRK2 expression. LRRK2 induced high expression of HMGB1 via P53. When blocking HMGB1 using the anti-HMGB1 antibody, the deteriorating effects caused by LRRK2 overexpression following hypoxia were inhibited in cardiomyocytes.Conclusions: In summary, LRRK2 deficiency protects hearts from myocardial infarction injury. The mechanism underlying this phenomenon involves the P53-HMGB1 pathway.