O-ring-induced transverse aortic constriction (OTAC) is a new simple method to develop cardiac hypertrophy and heart failure in mice

Suture-based transverse aortic constriction (TAC) in mice is one of the most frequently used experimental models for cardiac pressure overload-induced heart failure. However, the incidence of heart failure in the conventional TAC depends on the operator’s skill. To optimize and simplify this method, we proposed O-ring-induced transverse aortic constriction (OTAC) in mice. C57BL/6J mice were subjected to OTAC, in which an o-ring was applied to the transverse aorta (between the brachiocephalic artery and the left common carotid artery) and tied with a triple knot. We used different inner diameters of o-rings were 0.50 and 0.45 mm. Pressure overload by OTAC promoted left ventricular (LV) hypertrophy. OTAC also increased lung weight, indicating severe pulmonary congestion. Echocardiographic findings revealed that both OTAC groups developed LV hypertrophy within one week after the procedure and gradually reduced LV fractional shortening. In addition, significant elevations in gene expression related to heart failure, LV hypertrophy, and LV fibrosis were observed in the LV of OTAC mice. We demonstrated the OTAC method, which is a simple and effective cardiac pressure overload method in mice. This method will efficiently help us understand heart failure (HF) mechanisms with reduced LV ejection fraction (HFrEF) and cardiac hypertrophy.

Aerobic Exercise Attenuates Pressure Overload-Induced Cardiac Dysfunction through Promoting Skeletal Muscle Microcirculation and Increasing Muscle Mass

Background. Aerobic exercise has been proven to have a positive effect on cardiac function after hypertension; however, the mechanism is not entirely clarified. Skeletal muscle mass and microcirculation are closely associated with blood pressure and cardiac function. Objective. This study was designed to investigate the effects of aerobic exercise on the skeletal muscle capillary and muscle mass, to explore the possible mechanisms involved in exercise-induced mitigation of cardiac dysfunction in pressure overload mice. Methods. In this study, 60 BALB/C mice aged 8 weeks were randomly divided into 3 groups: control (CON), TAC, and TAC plus exercise (TAE) group and utilized transverse aortic constriction (TAC) to establish hypertensive model; meanwhile, treadmill training is used for aerobic exercise. After 5 days of recovery, mice in the TAE group were subjected to 10-week aerobic exercise. Carotid pressure and cardiac function were examined before mice were executed by Millar catheter and ultrasound, respectively. Muscle mass of gastrocnemius was weighed; cross-sectional area and the number of capillaries of gastrocnemius were detected by HE and immunohistochemistry, respectively. The mRNA and protein levels of VEGF in skeletal muscle were determined by RT-PCR and western blot, respectively. Results. We found that ① 10-week aerobic exercise counteracted hypertension and attenuated cardiac dysfunction in TAC-induced hypertensive mice; ② TAC decreased muscle mass of gastrocnemius and resulted in muscle atrophy, while 10-week aerobic exercise could reserve transverse aortic constriction-induced the decline of muscle mass and muscle atrophy; and ③ TAC reduced the number of capillaries and the protein level of VEGF in gastrocnemius, whereas 10-week aerobic exercise augmented the number of capillaries, the mRNA and protein levels of VEGF in mice were subjected to TAC surgery. Conclusions. This study indicates that 10-week aerobic exercise might fulfill its blood pressure-lowering effect via improving skeletal muscle microcirculation and increasing muscle mass.

Synus node dysfunction in heart failure is characterized by reduced CaMKII signaling

Heart failure (HF) is a complex syndrome in which death rates are over 50%. The main cause of death among HF patients is pump failure and ventricular arrhythmias, but severe bradycardia is also a common cause of sudden cardiac death, pointing to sinoatrial node (SAN) dysfunction. SAN pacemaker activity is regulated by voltage-clock and Ca2+-clock mechanisms and, although voltage-clock dysfunction in SAN has been largely proved in HF, Ca2+-clock dysfunction mechanisms in SAN remains undiscovered. Here, we used a HF model in mice with transverse aortic constriction (TAC) and using telemetry saw slower heart rhythm under autonomic nervous system blockade. Then, using confocal microscopy we analyzed Ca2+ handling in HF SAN tissue and found that intracellular Ca2+ transient rates were slower in addition to less frequency of Ca2+ sparks than in SHAM SAN tissue. Next, we studied protein expression of key excitation–contraction coupling proteins and found reduced expression of the Na+/Ca2+ exchanger and reduced phosphorylated status of ryanodine receptor and phospholamban in the CaMKII sites for the SAN in TAC mice. Finally, the application of the CaMKII inhibitor KN93 caused less effect in slowing the Ca2+ transient rates in HF SAN tissue, confirming the reduced CaMKII activation. In conclusion, our data demonstrate a reduction in CaMKII activation in the Ca2+-clock function of the SAN tissue in a mouse model of HF.

Synus node dysfunction in heart failure is characterized by reduced CaMKII signaling

Heart failure (HF) is a complex syndrome in which death rates are >50%. The main causes of death among HF patients are pump failure and ventricular arrhythmias, but severe bradycardia is also a common cause of sudden cardiac death, pointing to sinoatrial node (SAN) dysfunction. SAN pacemaker activity is regulated by voltage-clock and Ca2+-clock mechanisms and, although voltage-clock dysfunction in SAN has been largely proved in HF, Ca2+-clock dysfunction mechanisms in SAN remains unraveled. Here, we used an HF model in mice with transverse aortic constriction (TAC) and, using telemetry, saw slower heart rhythm under autonomic nervous system blockade. Then, by confocal microscopy, we analyzed Ca2+ handling in HF SAN tissue and found that intracellular Ca2+ transients rate were slower together with less frequency of Ca2+ sparks than in SHAM SAN tissue. Next, we studied protein expression of key excitation–contraction coupling proteins and found reduced expression of the Na+/Ca2+ exchanger and reduced phosphorylated status of ryanodine receptor and phospholamban in the CaMKII sites for the SAN in TAC mice. Finally, the application of the CaMKII inhibitor, KN93, caused less effect in slowing the Ca2+ transient rates in HF SAN tissue, confirming the reduced CaMKII activation. In conclusion, our data demonstrates a reduction in CaMKII activation in the Ca2+-clock function of the SAN tissue in a mouse model of HF.

Cardiac expression of microRNA-7 is associated with adverse cardiac remodeling

Although microRNA-7 (miRNA-7) is known to regulate proliferation of cancer cells by targeting Epidermal growth factor receptor (EGFR/ERBB) family, less is known about its role in cardiac physiology. Transgenic (Tg) mouse with cardiomyocyte-specific overexpression of miRNA-7 was generated to determine its role in cardiac physiology and pathology. Echocardiography on the miRNA-7 Tg mice showed cardiac dilation instead of age-associated physiological cardiac hypertrophy observed in non-Tg control mice. Subjecting miRNA-7 Tg mice to transverse aortic constriction (TAC) resulted in cardiac dilation associated with increased fibrosis bypassing the adaptive cardiac hypertrophic response to TAC. miRNA-7 expression in cardiomyocytes resulted in significant loss of ERBB2 expression with no changes in ERBB1 (EGFR). Cardiac proteomics in the miRNA-7 Tg mice showed significant reduction in mitochondrial membrane structural proteins compared to NTg reflecting role of miRNA-7 beyond the regulation of EGFR/ERRB in mediating cardiac dilation. Consistently, electron microscopy showed that miRNA-7 Tg hearts had disorganized rounded mitochondria that was associated with mitochondrial dysfunction. These findings show that expression of miRNA-7 in the cardiomyocytes results in cardiac dilation instead of adaptive hypertrophic response during aging or to TAC providing insights on yet to be understood role of miRNA-7 in cardiac function.

G Protein–Coupled Estrogen Receptor 30 Reduces Transverse Aortic Constriction–Induced Myocardial Fibrosis in Aged Female Mice by Inhibiting the ERK1/2 -MMP-9 Signaling Pathway

The incidence of cardiovascular diseases was significantly increased in postmenopausal women. The protection of estrogen in the cardiovascular system has been further reported for decades. Although menopausal hormone therapy has been used in many clinical trials, the debatable results indicate that the studies for elucidating the precise molecular mechanism are urgently required. G protein–coupled estrogen receptor 30 (GPR30) is a membrane receptor of estrogen and displays protective roles in diverse cardiovascular diseases. Previous studies have revealed that ERK1/2-mediated MMP-9 signaling was involved in ischemic heart diseases. However, the role of ERK1/2-mediated MMP-9 signaling in the protection of GPR30 against cardiac hypertrophy in aged female mice has not been investigated. Our present study demonstrated that GPR30 overexpression and its agonist G1 co-administration reduced transverse aortic constriction–induced myocardial fibrosis and preserved cardiac function in aged female mice. MMP-9 expression was markedly increased via ERK1/2 phosphorylation in transverse aortic constriction–injured myocardium of aged female mice. Further results showed that GPR30/G1 activation decreased MMP-9 expression via ERK1/2 inhibition, which further reduced TGF-β1 expression. Inhibition of the ERK1/2 signaling pathway by its inhibitor PD98059 suppressed the induction of the cardiomyocyte MMP-9 level caused by the GRP30 antagonist G15 and inhibited TGF-β1 expression in cardiac fibroblast in vitro. In summary, our results from in vivo and in vitro studies indicated that GPR30 activation inhibited myocardial fibrosis and preserved cardiac function via inhibiting ERK-mediated MMP-9 expression. Thus, the present study may provide the novel drug targets for prevention and treatment of cardiac pathological hypertrophy in postmenopausal women.

Characterization of Transverse Aortic Constriction in Mice Based on the Specific Recruitment of Leukocytes to the Hypertrophic Myocardium and the Aorta Ascendens

Transverse aortic constriction (TAC) is a model that mimics pressure overload-induced left ventricular (LV) hypertrophy in mice. Alterations in immune cell functionality can promote cardiac and vascular remodeling. In the present study, we characterized the time course in innate immune cell dynamics in response to TAC in the different tissues of mice. It was determined whether TAC induces a characteristic leukocyte-driven immune response in the myocardium, aorta ascendens and descendens, spleen, blood, and draining lymph nodes supported by cytokine-driven chemotaxis in mice at 3, 6, and 21 days following surgery. We used complex flow cytometry staining combinations to characterize the various innate immune cell subsets and a multiplex array to determine cytokine concentrations in the serum. The results of the current study indicated that leukocytes accumulate in the myocardium and aorta ascendens in response to TAC. The leukocyte dynamics in the myocardium were dominated by the Ly6Clow macrophages with an early accumulation, whereas the response in the aorta ascendens was characterized by a long-lasting proinflammatory phenotype driven by Ly6Chigh macrophages, neutrophils, and activated DCs. In contrast to the high-pressure environment of the aorta ascendens, the tissue of the aorta descendens did not react to TAC with any leukocyte increase. The levels of proinflammatory cytokines in the blood were elevated in response to TAC, indicating a systemic reaction. Moreover, our findings strongly suggest that cardiac macrophages could origin from splenic pools and reach the site of the inflammation via the blood. Based on the current findings, it can be concluded that the high-pressure conditions in the aorta ascendens cause a characteristic immune response, dominated by the accumulation of leukocytes and the activation of DCs that varies in comparison to the immune cell dynamics in the myocardium and the aorta descendens.