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Increased matrix metalloprotease 9 (MMP9) after myocardial infarction (MI) exacerbates ischemia-induced chronic heart failure (CHF). Autophagy is cardioprotective during CHF; however, whether increased MMP9 suppresses autophagic activity in CHF is unknown. This study aimed to determine whether increased MMP9 suppressed autophagic flux and MMP9 inhibition increased autophagic flux in the heart of rats with post-MI CHF. Sprague-Dawley rats underwent either sham surgery or coronary artery ligation 6–8 wk before being treated with MMP9 inhibitor for 7 days, followed by cardiac autophagic flux measurement with lysosomal inhibitor bafilomycin A1. Furthermore, autophagic flux was measured in vitro by treating H9c2 cardiomyocytes with two independent pharmacological MMP9 inhibitors, salvianolic acid B (SalB) and MMP9 inhibitor-I, and CRISPR/cas9-mediated MMP9 genetic ablation. CHF rats showed cardiac infarct, significantly increased left ventricular end-diastolic pressure (LVEDP), and increased MMP9 activity and fibrosis in the peri-infarct areas of left ventricular myocardium. Measurement of the autophagic markers LC3B-II and p62 with lysosomal inhibition showed decreased autophagic flux in the peri-infarct myocardium. Treatment with SalB for 7 days in CHF rats decreased MMP9 activity and cardiac fibrosis but increased autophagic flux in the peri-infarct myocardium. As an in vitro corollary study, measurement of autophagic flux in H9c2 cardiomyocytes and fibroblasts showed that pharmacological inhibition or genetic ablation of MMP9 upregulates autophagic flux. These data are consistent with our observations that MMP9 inhibition upregulates autophagic flux in the heart of rats with CHF. In conclusion, the results in this study suggest that the beneficial outcome of MMP9 inhibition in pathological cardiac remodeling is in part mediated by improved autophagic flux. NEW & NOTEWORTHY This study elucidates that the improved cardiac extracellular matrix (ECM) remodeling and cardioprotective effect of matrix metalloprotease 9 (MMP9) inhibition in chronic heart failure (CHF) are via increased autophagic flux. Autophagy is cardioprotective; however, the mechanism of autophagy suppression in CHF is unknown. We for the first time demonstrated here that increased MMP9 suppressed cardiac autophagy and ablation of MMP9 increased cardiac autophagic flux in CHF rats. Restoring the physiological level of autophagy in the failing heart is a challenge, and our study addressed this challenge. The novelty and highlights of this report are as follows: 1) MMP9 regulates cardiomyocyte and fibroblast autophagy, 2) MMP9 inhibition protects CHF after myocardial infarction (MI) via increased cardiac autophagic flux, 3) MMP9 inhibition increased cardiac autophagy via activation of AMP-activated protein kinase (AMPK)α, Beclin-1, Atg7 pathway and suppressed mechanistic target of rapamycin (mTOR) pathway.

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.

Introduction: Diastolic dysfunction and cardiac hypertrophic remodeling are hallmark features of heart failure with preserved ejection fraction (HFpEF) in humans. However, an ideal animal model that mimics all these basic clinical features of HFpEF remains to be developed. Methods: Male Sprague-Dawley rats (250-300 g) were subjected to central infusion of either Angiotensin II (Ang II at 20 ng/min, 0.5 μl/h, i.c.v) or isotonic saline (0.5 μl/h, i.c.v) through osmotic mini-pumps for 14 days. Echocardiography was used to measure systolic (ejection fraction) and diastolic functions (E/A and E/E’ ratio) at day 14. Mean arterial pressure (MAP), heart rate (HR), left ventricular pressure (LVP), changes in ±dP/dt, and cardiac responses to isoproterenol (ISO) were also measured in anesthetized rats. Results: Central infusion of Ang II in rats resulted in increased sympathoexcitation and mean arterial pressure. Concomitantly, there was decreased E/A ratio (AngII: 1.2 ± 0.1 vs. Cont: 1.5 ± 0.1) and increased E/E’ ratio (AngII: 14.3 ± 0.8 vs. Cont: 12.9 ± 0.6), indicating diastolic dysfunction. Ejection fractions were comparable between the groups (AngII: 67 ± 2% vs. Cont: 69 ± 5%). Ang II infused rats demonstrated increased heart to body weight ratio (AngII: 4.5 ± 0.5 vs. Cont: 2.9 ± 0.1), cachexia (AngII: 235 ± 18 g vs. Cont: 331 ± 11 g), hypertrophy (AngII: 46 ± 5 μm/unit area vs. Cont: 36 ± 7 μm/unit area), and fibrosis (AngII: 5.2 ± 0.5 intensity/unit area vs. Cont: 2.6 ± 0.4 intensity/unit area). At the same time cardiac contractile responsiveness to ISO (0.5 μg/kg) was significantly lower for negative dP/dt in Ang II rats (AngII: -6998 mmHg/s vs. Cont: -11675 mmHg/s), which is indicative of abnormal diastole or relaxation. However, positive dP/dt response to ISO was similar in the two groups, indicating preserved systolic function in the Ang II group. Conclusions: Central Ang II infusion in rats shows sympathoexcitation, diastolic dysfunction, and pathological cardiac hypertrophy and fibrosis mimicking abnormalities seen in patients with HFpEF. This model of cardiac dysfunction in rats may represent a suitable platform to investigate the underlying mechanisms of HFpEF.

BackgroundGlucagon-like peptide-1 (GLP-1) induces diuresis and natriuresis. Previously we have shown that GLP-1 activates afferent renal nerve to increase efferent renal sympathetic nerve activity that negates the diuresis and natriuresis as a negative feedback mechanism in normal rats. However, renal effects of GLP-1 in heart failure (HF) has not been elucidated. The present study was designed to assess GLP-1-induced diuresis and natriuresis in rats with HF and its interactions with renal nerve activity.MethodsHF was induced in rats by coronary artery ligation. The direct recording of afferent renal nerve activity (ARNA) with intrapelvic injection of GLP-1 and total renal sympathetic nerve activity (RSNA) with intravenous infusion of GLP-1 were performed. GLP-1 receptor expression in renal pelvis, densely innervated by afferent renal nerve, was assessed by real-time PCR and western blot analysis. In separate group of rats after coronary artery ligation selective afferent renal denervation (A-RDN) was performed by periaxonal application of capsaicin, then intravenous infusion of GLP-1-induced diuresis and natriuresis were evaluated.ResultsIn HF, compared to sham-operated control; (1) response of increase in ARNA to intrapelvic injection of GLP-1 was enhanced (3.7 ± 0.4 vs. 2.0 ± 0.4 µV s), (2) GLP-1 receptor expression was increased in renal pelvis, (3) response of increase in RSNA to intravenous infusion of GLP-1 was enhanced (132 ± 30% vs. 70 ± 16% of the baseline level), and (4) diuretic and natriuretic responses to intravenous infusion of GLP-1 were blunted (urine flow 53.4 ± 4.3 vs. 78.6 ± 4.4 µl/min/gkw, sodium excretion 7.4 ± 0.8 vs. 10.9 ± 1.0 µEq/min/gkw). A-RDN induced significant increases in diuretic and natriuretic responses to GLP-1 in HF (urine flow 96.0 ± 1.9 vs. 53.4 ± 4.3 µl/min/gkw, sodium excretion 13.6 ± 1.4 vs. 7.4 ± 0.8 µEq/min/gkw).ConclusionsThe excessive activation of neural circuitry involving afferent and efferent renal nerves suppresses diuretic and natriuretic responses to GLP-1 in HF. These pathophysiological responses to GLP-1 might be involved in the interaction between incretin-based medicines and established HF condition. RDN restores diuretic and natriuretic effects of GLP-1 and thus has potential beneficial therapeutic implication for diabetic HF patients.

Diabetic cardiomyopathy is a leading cause of heart failure. Developing a novel therapeutic strategy for diabetic cardiomyopathy and characterizing animal models used for diabetes mellitus (DM) are important. Insulin 2 mutant (Ins2+/-) Akita is a spontaneous, genetic, mouse model for T1DM, which is relevant to humans. There are contrasting reports on systolic dysfunction and pathological remodeling (hypertrophy and fibrosis) in Akita heart. Here, we used magnetic resonance imaging (MRI) approach, a gold standard reference for evaluating cardiac function, to measure ejection fraction (indicator of systolic dysfunction) in Akita. Moreover, we performed Wheat Germ Agglutinin (WGA) and hematoxylin and Eosin stainings to determine cardiac hypertrophy, and Masson's Trichrome and picrosirius red stainings to determine cardiac fibrosis in Akita. MiR-133a, an anti-hypertrophy and anti-fibrosis miRNA, is downregulated in Akita heart. We determined if miR-133a mimic treatment could mitigate systolic dysfunction and remodeling in Akita heart. Our MRI results revealed decreased ejection fraction in Akita as compared to WT and increased ejection fraction in miR-133a mimic-treated Akita. We also found that miR-133a mimic treatment mitigates T1DM-induced cardiac hypertrophy and fibrosis in Akita. We conclude that Akita shows cardiac hypertrophy, fibrosis and systolic dysfunction and miR-133a mimic treatment to Akita could ameliorate them.

An activated renin-angiotensin system (RAS) within the central nervous system has been implicated in sympathoexcitation during various disease conditions including congestive heart failure (CHF). In particular, activation of the RAS in the paraventricular nucleus (PVN) of the hypothalamus has been recognized to augment sympathoexcitation in CHF. We observed a 2.6-fold increase in angiotensinogen (AGT) in the PVN of CHF. To elucidate the molecular mechanism for increased expression of AGT, we performed in silico analysis of the 3′-untranslated region (3′-UTR) of AGT and found a potential binding site for microRNA (miR)-133a. We hypothesized that decreased miR-133a might contribute to increased AGT in the PVN of CHF rats. Overexpression of miR-133a in NG108 cells resulted in 1.4- and 1.5-fold decreases in AGT and angiotensin type II (ANG II) type 1 receptor (AT1R) mRNA levels, respectively. A luciferase reporter assay performed on NG108 cells confirmed miR-133a binding to the 3′-UTR of AGT. Consistent with these in vitro data, we observed a 1.9-fold decrease in miR-133a expression with a concomitant increase in AGT and AT1R expression within the PVN of CHF rats. Furthermore, restoring the levels of miR-133a within the PVN of CHF rats with viral transduction resulted in a significant reduction of AGT (1.4-fold) and AT1R (1.5-fold) levels with a concomitant decrease in basal renal sympathetic nerve activity (RSNA). Restoration of miR-133a also abrogated the enhanced RSNA responses to microinjected ANG II within the PVN of CHF rats. These results reveal a novel and potentially unique role for miR-133a in the regulation of ANG II within the PVN of CHF rats, which may potentially contribute to the commonly observed sympathoexcitation in CHF.NEW & NOTEWORTHY Angiotensinogen (AGT) expression is upregulated in the paraventricular nucleus of the hypothalamus through posttranscriptional mechanism interceded by microRNA-133a in heart failure. Understanding the mechanism of increased expression of AGT in pathological conditions leading to increased sympathoexcitation may provide the basis for the possible development of new therapeutic agents with enhanced specificity.

Activation of renin-angiotensin system (RAS) within the paraventricular nucleus (PVN) of the hypothalamus has been suggested to enhance sympathetic activation in chronic heart failure (CHF) condition. The contribution due to changes in angiotensinogen (AGT) per se remains to be examined. In this study, we observed a 12-fold increase in levels of AGT gene in the PVN of CHF rats compared to sham-operated controls. In order to elucidate the molecular mechanism for increased expression of AGT, we performed in silico analysis of 3'-UTR of AGT gene and found potential binding site for microRNA (miR)-133a. We found a 1.9-fold decrease in mir-133a expression in the PVN of rats with CHF. We hypothesized that decreased expression of mir-133a may be responsible for the increased expression of AGT in the PVN. To test this hypothesis we used NG108 (neuroblastoma x glioma) cell line, which endogenously expresses RAS, as our in vitro model. Overexpression of miR133a (pEZX-MR03-miR133a) in NG108 cells resulted in 1.4 and...

Although Mdivi-1 ameliorates heart failure by inhibiting autophagy, the mechanism of Mdivi-1 mediated mitigation of cardiac dysfunction in diabetes is unclear. We hypothesize that Mdivi-1 improves ...

Diabetes is a rapidly increasing disease that enhances the chances of heart failure twofold to fourfold (as compared to age and sex matched nondiabetics) and becomes a leading cause of morbidity and mortality. There are two broad classifications of diabetes: type1 diabetes (T1D) and type2 diabetes (T2D). Several mice models mimic both T1D and T2D in humans. However, the genetic intervention to ameliorate diabetic cardiomyopathy in these mice often requires creating double knockout (DKO). In order to assess the therapeutic potential of a gene, that specific gene is either overexpressed (transgenic expression) or abrogated (knockout) in the diabetic mice. If the genetic mice model for diabetes is used, it is necessary to create DKO with transgenic/knockout of the target gene to investigate the specific role of that gene in pathological cardiac remodeling in diabetics. One of the important genes involved in extracellular matrix (ECM) remodeling in diabetes is matrix metalloproteinase-9 (Mmp9). Mmp9 is a collagenase that remains latent in healthy hearts but induced in diabetic hearts. Activated Mmp9 degrades extracellular matrix (ECM) and increases matrix turnover causing cardiac fibrosis that leads to heart failure. Insulin2 mutant (Ins2+/-) Akita is a genetic model for T1D that becomes diabetic spontaneously at the age of 3-4 weeks and show robust hyperglycemia at the age of 10-12 weeks. It is a chronic model of T1D. In Ins2+/- Akita, Mmp9 is induced. To investigate the specific role of Mmp9 in diabetic hearts, it is necessary to create diabetic mice where Mmp9 gene is deleted. Here, we describe the method to generate Ins2+/-/Mmp9-/- (DKO) mice to determine whether the abrogation of Mmp9 ameliorates diabetic cardiomyopathy.