Sleep deprivation, shift work, and jet lag all disrupt normal biological rhythms and have major impacts on health; however, circadian disorganization has never been shown as a causal risk factor in organ disease. We now demonstrate devastating effects of rhythm disorganization on cardiovascular and renal integrity and that interventions based on circadian principles prevent disease pathology caused by a short-period mutation (tau) of the circadian system in hamsters. The point mutation in the circadian regulatory gene, casein kinase-1epsilon, produces early onset circadian entrainment with fragmented patterns of behavior in +/tau heterozygotes. Animals die at a younger age with cardiomyopathy, extensive fibrosis, and severely impaired contractility; they also have severe renal disease with proteinuria, tubular dilation, and cellular apoptosis. On light cycles appropriate for their genotype (22 h), cyclic behavioral patterns are normalized, cardiorenal phenotype is reversed, and hearts and kidneys show normal structure and function. Moreover, hypertrophy does not develop in animals whose suprachiasmatic nucleus was ablated as young adults. Circadian organization therefore is critical for normal health and longevity, whereas chronic global asynchrony is implicated in the etiology of cardiac and renal disease.
Objective: The renin-angiotensin system (RAS) has been known for more than a century as a cascade that regulates body fluid balance, renal functions and blood pressure. Angiotensin-converting enzyme 2 (ACE2) is now known as a negative regulator of RAS, and activation of the ACE2 is a possible alternative target for new drugs, since some protective influences on renal and cardiovascular function have been revealed. We hypothesized that ACE2 would exert beneficial effects on oxidative stress levels and renal injury in apolipoprotein E (ApoE) -knockout (KO) mice. Design and method: In this study, we used 12-week-old wild-type, ApoEKO, and ACE2/ApoE double KO mice. The ApoEKO mice were treated with recombinant human ACE2 (hrACE2) with the daily dose of 2 mg/kg. We characterized the functional, structural and molecular signaling changes in mice kidneys. Results: Compared with the ApoEKO mice, ACE2 deficiency led to greater increases in renal oxidative stress levels and expression of oxidative stress-inducible proteins NADPH oxidase 4 (NOX4) in the ACE2/ApoE double KO mice. These changes were associated with exacerbation of renal tubule ultrastructure injury and greater activation of Akt and ERK1/2 phosphorylated signaling. Conversely, treatment with hrACE2 significantly attenuated renal oxidative stress levels and ultrastructure injury, and prevented the expression of NOX4 and phosphorylated level of Akt and ERK1/2 in ApoEKO mouse kidneys. However, there were no changes in renal expression of NOX2 and Mas receptor among groups. Conclusions: Deletion of ACE2 triggers greater increases in renal oxidative stress and tubular ultrastructure injury in the ACE2/ApoE double mutant mice with greater activation of Akt-ERK1/2 phosphorylated signaling. While ACE2 overexpression alleviates renal tubular injury in ApoE-mutant mice with suppression of superoxide generation and downregulation of the Akt-ERK phosphorylated signaling. Strategies aimed at enhancing ACE2 action may have important therapeutic potential for atherosclerosis and renal diseases.
Heart failure remains a highly prevalent condition with diverse etiology, yet the underlying signaling mechanisms are not fully understood. Despite the profound effects of post-translational protein modifications on downstream signaling, limited studies have investigated the cardiac phosphoproteome in human heart failure. We hypothesized that a combined proteomic and phosphoproteomic analysis of human dilated (DCM) and ischemic (ICM) cardiomyopathy would reveal novel etiology-associated disease pathways. Integrative analyses of left ventricular explants from DCM patients ( n =4) vs. non-failing controls ( n =4), and left ventricular infarct vs. non-infarct, and peri-infarct vs. non-infarct regions of ICM patients ( n =4) identified 5,570 unique proteins with 13,624 corresponding phosphorylation sites. Each pair-wise comparison revealed shared and etiology-specific signatures, with a unique DCM-associated enrichment of cell-cell adhesion pathways. We focused our attention on αT-catenin (CTNNA3) as a cardiomyocyte intercalated disc candidate phosphoprotein with a unique cluster of 4 hyperphosphorylated sites in DCM hearts ( P <0.0001). Overexpression of non-phosphorylatable hCTNNA3 in ex vivo isolated adult mouse cardiomyocytes showed internalized protein expression and weaker cell-cell adhesion vs. wildtype (WT) and phospho-mimetic forms. We established an in vivo mouse model using recombinant adeno-associated virus 9 (rAAV9) harboring hCTNNA3-WT, hCTNNA3-phospho-null, or empty rAAV9 control. Phospho-null CTNNA3 mice developed left ventricular dilation and contractile dysfunction (% EF; 51.25±1.17 phospho-null vs. 62.07±1.20 WT vs. 66.76±1.42 empty; n ≥10) with impaired left ventricular conduction velocity (cm/s; 36.83±1.10 phospho-null vs. 47.74±2.04 WT; n =6), by echocardiography and ex vivo optical mapping. Loss of CTNNA3 phosphorylation led to intercalated disc remodeling with internalization and dissociation of CTNNA3, connexin 43, N-cadherin, β-catenin, and plakophilin 2 from the adherens junction, using high-resolution confocal imaging. Together these findings reveal a compensatory role for αT-catenin phosphorylation in maintaining cardiomyocyte intercalated disc organization in human DCM.
Obesity is increasing in prevalence and is strongly associated with metabolic and cardiovascular disorders. The renin-angiotensin system (RAS) has emerged as a key pathogenic mechanism for these disorders; angiotensin (Ang)-converting enzyme 2 (ACE2) negatively regulates RAS by metabolizing Ang II into Ang 1-7. We studied the role of ACE2 in obesity-mediated cardiac dysfunction. ACE2 null (ACE2KO) and wild-type (WT) mice were fed a high-fat diet (HFD) or a control diet and studied at 6 months of age. Loss of ACE2 resulted in decreased weight gain but increased glucose intolerance, epicardial adipose tissue (EAT) inflammation, and polarization of macrophages into a proinflammatory phenotype in response to HFD. Similarly, human EAT in patients with obesity and heart failure displayed a proinflammatory macrophage phenotype. Exacerbated EAT inflammation in ACE2KO-HFD mice was associated with decreased myocardial adiponectin, decreased phosphorylation of AMPK, increased cardiac steatosis and lipotoxicity, and myocardial insulin resistance, which worsened heart function. Ang 1-7 (24 µg/kg/h) administered to ACE2KO-HFD mice resulted in ameliorated EAT inflammation and reduced cardiac steatosis and lipotoxicity, resulting in normalization of heart failure. In conclusion, ACE2 plays a novel role in heart disease associated with obesity wherein ACE2 negatively regulates obesity-induced EAT inflammation and cardiac insulin resistance.
Angiotensin-converting enzyme 2 (ACE2), the receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is highly expressed in the kidneys. Beyond serving as a crucial endogenous regulator of the renin-angiotensin system, ACE2 also possess a unique function to facilitate amino acid absorption. Our observational study sought to explore the relationship between urine ACE2 (uACE2) and renal outcomes in coronavirus disease 2019 (COVID-19).In a cohort of 104 patients with COVID-19 without acute kidney injury (AKI), 43 patients with COVID-19-mediated AKI and 36 non-COVID-19 controls, we measured uACE2, urine tumour necrosis factor receptors I and II (uTNF-RI and uTNF-RII) and neutrophil gelatinase-associated lipocalin (uNGAL). We also assessed ACE2 staining in autopsy kidney samples and generated a propensity score-matched subgroup of patients to perform a targeted urine metabolomic study to describe the characteristic signature of COVID-19.uACE2 is increased in patients with COVID-19 and further increased in those that developed AKI. After adjusting uACE2 levels for age, sex and previous comorbidities, increased uACE2 was independently associated with a >3-fold higher risk of developing AKI [odds ratio 3.05 (95% confidence interval 1.23‒7.58), P = .017]. Increased uACE2 corresponded to a tubular loss of ACE2 in kidney sections and strongly correlated with uTNF-RI and uTNF-RII. Urine quantitative metabolome analysis revealed an increased excretion of essential amino acids in patients with COVID-19, including leucine, isoleucine, tryptophan and phenylalanine. Additionally, a strong correlation was observed between urine amino acids and uACE2.Elevated uACE2 is related to AKI in patients with COVID-19. The loss of tubular ACE2 during SARS-CoV-2 infection demonstrates a potential link between aminoaciduria and proximal tubular injury.
Although angiotensin II (Ang II) plays an important role in heart disease associated with pump dysfunction, its direct effects on cardiac pump function remain controversial. We found that after Ang II infusion, the developed pressure and +dP/dt(max) in isolated Langendorff-perfused mouse hearts showed a complex temporal response, with a rapid transient decrease followed by an increase above baseline. Similar time-dependent changes in cell shortening and L-type Ca(2+) currents were observed in isolated ventricular myocytes. Previous studies have established that Ang II signaling involves phosphoinositide 3-kinases (PI3K). Dominant-negative inhibition of PI3Kalpha in the myocardium selectively eliminated the rapid negative inotropic action of Ang II (inhibited by approximately 90%), whereas the loss of PI3Kgamma had no effect on the response to Ang II. Consistent with a link between PI3Kalpha and protein kinase C (PKC), PKC inhibition (with GF 109203X) reduced the negative inotropic effects of Ang II by approximately 50%. Although PI3Kalpha and PKC activities are associated with glycogen synthase kinase-3beta and NADPH oxidase, genetic ablation of either glycogen synthase kinase-3beta or p47(phox) (an essential subunit of NOX2-NADPH oxidase) had no effect on the inotropic actions of Ang II. Our results establish that Ang II has complex temporal effects on contractility and L-type Ca(2+) channels in normal mouse myocardium, with the negative inotropic effects requiring PI3Kalpha and PKC activities.
Abstract Cardiac output (CO), heart rate (HR), stroke volume (SV), dorsal aortic blood flow (DABF), dorsal aortic blood pressure (P DA ) and plasma electrolytes were monitored in stanniectomized and sham-operated freshwater eels over a 3-week period; branchial shunting and systemic resistance (R SYS ) were estimated. DABF was significantly reduced by 45% from 11·72±0·48 (control) to 6·55±0·41 ( n =6; day 21) ml.min −1 .kg −1 within 3 weeks after the removal of the corpuscles of Stannius. This large reduction in blood flow was due to a 25% decrease in CO and a 100% increase in estimated branchial shunting which preceded the fall in CO. CO was decreased from 16·07 ±0·31 (control) to 11·91 ±1 ( n =6; day 21) ml.min −1 .kg −1 through a reduction in SV; there was no significant change in HR. Estimated branchial shunting, a relative measure of branchial arterio-venous blood flow, corresponded to 2·53±0·18 ml.min −1 .kg −1 (control; n =12), which represents 16% of baseline CO. Ventral and dorsal aortic pulse flows also decreased following stanniectomy. The decrease in DABF occurred in conjunction with a reduction in P DA which was measured for 12 days in a separate group of eels. Baseline P DA (3·03 ±0·1 kPa) significantly decreased by 15% to 2·55 ±0·13 kPa 4 days after stanniectomy. However, this fall in P DA was transient and accompanied by an elevation in derived R SYS . These results support the hypothesis that the corpuscles of Stannius are closely linked to cardiovascular regulation in freshwater eels. Electrolyte changes (hypercalcemia, hypomagnesia, hyperkalemia and hyponatremia) were temporally coupled to the changes in blood flows. Impaired cardiovascular function and altered patterns of blood flow to osmoregulatory organs such as the gills, kidney and skin may have led to some or all of the electrolyte disturbances which followed stanniectomy. Journal of Endocrinology (1995) 145, 181–194
Systolic heart failure is a cardiac disease in which the contractile function of heart muscle becomes weakened and unable to pump blood with adequate force. As of yet, no existing positive inotrope is able to lower the mortality rate in systolic heart failure. Cardiac troponin complex (cTn) is a trimeric complex consisting of troponin C (cTnC), troponin I (cTnI) and troponin T (cTnT). cTnC regulates blood supply to the heart by turning muscle contraction on and off in a calcium-dependent manner. More precisely, the regulatory N-domain of cTnC (cNTnC) binds the switch region of cTnI to activate contraction. Drug-like molecules need to bind to this cNTnC-cTnI switch region interface and modulate the pumping activity of the heart. Our objective was to identify a small molecule that has the ability to stabilize the activated conformation of cTn by binding to this interface and thereby enhance cardiac muscle contraction.A cardiac troponin activator could potentially compensate the impaired contractile function of the heart in the treatment of systolic heart failure METHODS: A total of 47 new compounds were designed using existing knowledge of calcium sensitizers. The compounds were screened against a recombinantly purified cNTnC-cTnI chimeric construct, named gChimera, using nuclear magnetic resonance and fluorescence spectroscopy. We identified a novel small molecule cardiac troponin activator, RPI-194, and measured its binding affinity to recombinantly purified cNTnC and gChimera. We also investigated the activity of RPI-194 in rat skinned cardiac muscle trabeculae, isolated mouse cardiomyocytes, and isolated working mouse hearts. RPI-194 activity was also tested in skinned skeletal muscle fibers from rats as both cardiac and slow skeletal muscle share the same isoform of cTnC.We demonstrated that a small molecule troponin activator RPI-194 binds to gChimera with a KD of 12-24 µM and is able to stabilize activated cTn. It also slowed down the rate of calcium release from reconstituted cTn. RPI-194 showed increasing calcium sensitivity of isometric contraction in skinned cardiac muscle trabeculae, as well as in slow and fast skeletal muscle fibers, suggesting it to be a calcium sensitizer, with cross-reactivity within striated muscle. Contrarily, RPI-194 was unable to slow down calcium release from isolated cNTnC. It also reduced the velocity of unloaded shortening in skeletal muscle fibers, suggesting that it slows the rate of actin-myosin cross-bridging, although no effect on myosin ATPase activity was found. RPI-194 decreased the velocity and amplitude of contractions in isolated cardiomyocytes but contractility was preserved in isolated working hearts.RPI-194 is a small molecule troponin activator that acts as a calcium sensitizer in striated muscle. Because of the isoform sharing of cTnC in cardiac and slow skeletal muscle, it is nearly impossible to develop a cardiac-specific troponin activator. Whole animal model studies are needed to determine the impact of RPI-194 on the different striated muscle types and whether this would be acceptable for therapeutic intervention.
