Trehalose Ameliorates Diabetic Cardiomyopathy: Role of the PK2/PKR Pathway

Ample clinical case reports suggest a high incidence of cardiomyopathy in diabetes mellitus (DM). Recent evidence supports an essential role of trehalose (TLS) in cardiomyocyte survival signaling. Our previous study found that prokineticin2 (PK2) was involved in the process of diabetic cardiomyopathy (DCM). The present study examined the protective effects and mechanisms of TLS on DM-induced cardiomyocyte injury in mice and H9c2 cardiomyocytes. C57BL/6J mice were intraperitoneally injected with 50 mg·kg-1·d-1 streptozotocin for five consecutive days to establish an experimental diabetic model and then administered TLS (1 mg·g-1·d-1, i.p.) for two days every 4 weeks and given 2% TLS in drinking water for 24 weeks. Echocardiography, myocardial structure, apoptosis, pyroptosis, autophagy, and the PK2/PKR pathway were assessed. Cardiomyocytes exposed to high glucose (HG) were treated with TLS in the absence or presence of the PK2 antagonist PKRA7, and proteins involved in apoptosis, autophagy, and pyroptosis and the PK2/PKR pathways were evaluated using Western blot analysis. Diabetic mice demonstrated metabolic disorder, abnormal myocardial zymograms, and aberrant myocardial systolic and diastolic function, which were accompanied by pronounced apoptosis, pyroptosis, and dampened autophagy. TLS treatment relieved these effects. PK2 and receptor expressions were downregulated in diabetic mice, and TLS nullified this effect. PKRA7 eliminated the impact of TLS on cardiomyocytes. This evidence suggests that TLS rescues DM-induced myocardial function, pyroptosis, and apoptosis, likely via the PK2/PKR pathway.

Cpxm2 as a novel candidate for cardiac hypertrophy and failure in hypertension

Treatment of hypertension-mediated cardiac damage with left ventricular (LV) hypertrophy (LVH) and heart failure remains challenging. To identify novel targets, we performed comparative transcriptome analysis between genetic models derived from stroke-prone spontaneously hypertensive rats (SHRSP). Here, we identified carboxypeptidase X 2 (Cpxm2) as a genetic locus affecting LV mass. Analysis of isolated rat cardiomyocytes and cardiofibroblasts indicated Cpxm2 expression and intrinsic upregulation in genetic hypertension. Immunostaining indicated that CPXM2 associates with the t-tubule network of cardiomyocytes. The functional role of Cpxm2 was further investigated in Cpxm2-deficient (KO) and wild-type (WT) mice exposed to deoxycorticosterone acetate (DOCA). WT and KO animals developed severe and similar systolic hypertension in response to DOCA. WT mice developed severe LV damage, including increases in LV masses and diameters, impairment of LV systolic and diastolic function and reduced ejection fraction. These changes were significantly ameliorated or even normalized (i.e., ejection fraction) in KO-DOCA animals. LV transcriptome analysis showed a molecular cardiac hypertrophy/remodeling signature in WT but not KO mice with significant upregulation of 1234 transcripts, including Cpxm2, in response to DOCA. Analysis of endomyocardial biopsies from patients with cardiac hypertrophy indicated significant upregulation of CPXM2 expression. These data support further translational investigation of CPXM2.

MicroRNA-383-5p Regulates Oxidative Stress in Mice with Acute Myocardial Infarction through the AMPK Signaling Pathway via PFKM

Objective. The purpose of this study is to explore the regulating role of microRNA-383-5p (miR-383-5p) in oxidative stress after acute myocardial infarction (AMI) through AMPK pathway via phosphofructokinase muscle-type (PFKM). Methods. We established the AMI model, and the model mice were injected with miR-383-5p agomir to study the effect of miR-383-5p in AMPK signaling pathways. The target gene for miR-383-5p was reported to be PFKM, so we hypothesized that overexpression of miR-383-5p inhibits activation of the AMPK signaling pathway. Results. In this research, we found that overexpression of miR-383-5p decreases myocardial oxidative stress, myocardial apoptosis, the expression level of PFKM malondialdehyde (MDA), and reactive oxygen species (ROS) in the myocardial tissues after AMI, and finally, AMI-induced cardiac systolic and diastolic function could be improved.Conclusion. This study demonstrated that miR-383-5p could reduce the oxidative stress after AMI through AMPK signaling pathway by targeting PFKM.

586 Effect of sacubitril/valsartan combination in an experimental model of heart failure

Aims The majority of elderly patients with heart failure has a preserved ejection fraction (HFpEF) that constitutes a syndrome characterized by frequent hospitalizations and high mortality. Despite the growing social burden of HFpEF, the comprehension of its pathophysiology is incomplete, and treatment remains largely undefined. Ageing itself may contribute independently to deterioration of diastolic function. Methods and results An 18-month-old female Fischer 344 rats were treated with oral administration of either sacubitril/valsartan (60 mg/kg/die, 1:1 ratio) or valsartan alone (30 mg/kg/die) for 12 weeks. Tail-cuff method was used to monitor blood pressure weekly. Echocardiography and left ventricle catheterization were employed to assess systolic and diastolic function, at baseline, and before sacrifice. Cardiac tissue was used for molecular biology and histochemistry assays. Tail-cuff analysis indicated a comparable decrease in blood pressure between treatments. Hypertrophy also showed a significant reduction with both treatments. On the contrary, myocardial function analysis demonstrated that no treatment was efficacy on diastolic dysfunction. The lack of improvement of cardiac function could be attributed to the inability of the treatments to counteract the accumulation of fibrotic tissue in the left ventricle, which, in turn, is attributable to the failure to reduce the inflammatory process and oxidative stress, and to the inability to modulate angiotensin II pathway. Conclusions Our results evidenced that both sacubitril/valsartan or valsartan treatment was able to improve diastolic function and pro-fibrotic remodelling, partly due to a lack of effect on classical and non-classical pathways of angiotensin II.

Troponin I phosphorylation is essential for cardiac reserve

With an increase in the body’s metabolic demand (e.g., exercise), the heart must increase its pumping performance. To achieve this increased performance, the heart relies on its cardiac reserve, which is the ability to increase its systolic and diastolic function. The mechanism responsible for cardiac reserve is poorly understood. The myofilaments are essential for contraction/relaxation, with troponin I (the inhibitory subunit of troponin, TnI) being a key regulatory protein. Studies have shown that TnI serine 23/24 (S23/S24) phosphorylation is a key mechanism for accelerating relaxation by decreasing Ca2+ sensitivity. However, the role of TnI in cardiac reserve is unknown. For this study, we characterized the systolic and diastolic reserve in TnI S23/S24 phosphorylation-null transgenic mice (S23/S24 mutated to alanine [AA] mice). Even with increased Ca2+ sensitivity, the AA mice exhibited normal function at resting heart rate with no difference in cardiac structure compared with wild type. To examine the role TnI S23/S24 phosphorylation in systolic and diastolic reserve, we assessed hemodynamics via left ventricular catheterization on the Bowditch effect (i.e., an increase in contractile function with increasing heart rate) by increasing heart rate (from 240 to 420 beats per minute) and sympathetic stimulation (dobutamine). Our data exhibited a clear loss of diastolic and systolic reserve in the AA mice with increasing heart rate and dobutamine. Since we observed a clear inability to increase systolic and diastolic function in AA mice, we performed speckle tracking echocardiography to quantitatively characterize function at resting heart rate. We observed that AA mice demonstrated normal systolic function (radial strain rate) and impaired directional diastolic function (reverse radial strain rate) at resting heart rate. We conclude that TnI S23/S24 phosphorylation is essential for cardiac reserve by enhancing systolic and diastolic function. A blunted cardiac reserve leads to heart disease making TnI S23/S24 phosphorylation a potential therapeutic strategy.

Compound Mutation in Cardiac Sarcomere Proteins Is Associated with Increased Risk for Major Arrhythmic Events in Pediatric Onset Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is associated with adverse left ventricular (LV) remodeling causing dysfunction and malignant arrhythmias. Severely affected patients present with disease onset during childhood and sudden cardiac death risk (SCD) stratification is of the highest importance in this cohort. This study aimed to investigate genotype–phenotype association regarding clinical outcome and disease progression in pediatric onset HCM. Medical charts from forty-nine patients with pediatric HCM who had undergone genetic testing were reviewed for retrospective analysis. Demographic, clinical, transthoracic echocardiographic, electrocardiographic, long-term electrocardiogram, cardiopulmonary exercise test, cardiac magnetic resonance, and medication data were recorded. Childhood onset HCM was diagnosed in 29 males and 20 females. Median age at last follow-up was 18.7 years (range 2.6–51.7 years) with a median follow-up time since diagnosis of 8.5 years (range 0.2–38.0 years). Comparison of patients carrying mutations in distinct genes and comparison of genotype-negative with genotype-positive individuals, revealed no differences in functional classification, LV morphology, hypertrophy, systolic and diastolic function, fibrosis and cardiac medication. Patients with compound mutations had a significantly higher risk for major arrhythmic events than a single-mutation carrier. No association between affected genes and disease severity or progression was identified in this cohort.

Assessment of Cardiac Abnormalities in Sickle Cell Disease Patients Using Cardiac Magnetic Resonance Imaging (CMR)

Introduction Sickle cell disease (SCD) is characterized by chronic hemolytic anemia which predisposes patients to high output heart failure. Recently it has been recognized that repetitive microvascular ischemic insults and reperfusion injury result in diffuse myocardial fibrosis and cardiac remodeling that eventually leads to irreparable myocardial injury. Superimposed on these cardiac insults, patients with SCD often have iron overload related to chronic transfusions. Together, these insults produce a unique SCD-related cardiomyopathy characterized by restrictive physiology with LV dilation. Cardiac Magnetic Resonance Imaging (CMR) is the only non-invasive technique for assessing and quantifying diffuse myocardial fibrosis with simultaneous highly accurate assessment of heart size and systolic and diastolic function. Diffuse myocardial fibrosis is measured by a CMR technique called T1 mapping and calculated as extracellular volume (ECV), which may serve as an early marker of sickle cell related cardiomyopathy. An early marker for SCD-related cardiomyopathy would allow for efficient screening, prevention, and institution of treatment strategies. We describe utilization of CMR to evaluate cardiac pathology in a cohort of pediatric SCD patients. Methods Twenty-six children with SCD underwent CMR, as clinically indicated, between January 2020 and July 2021 at our institution. We analyzed cardiac chamber size (left atrium [LA], left ventricle [LV], right ventricle [RV]), systolic and diastolic function parameters, and quantitative myocardial parametric mapping values (T1 pre- and post-contrast and T2*) for these patients. ECV was calculated for all patients using T1 pre- and post-contrast values in conjunction with current hemoglobin values. Four patients underwent an allogeneic hematopoietic cell transplant (HCT) for SCD and had serial CMR performed before and 1 month after HCT. Results Median age of patients was 17 years; 12 (70%) were female; 22 (84%) had HbSS, 3 (12%) had HbSC and 1 (4%) had Hb S beta thalassemia. 15 (58%) patients had LA enlargement and only a few had ventricular enlargement (LV, 11 [42%]; RV, 9 [35%]). Patients rarely had had LV (4 [15%]) or RV (1 [4%]) systolic dysfunction. Twenty-five (96%) patients had increased ECV; median 30.5% (IQR 28-34%, reference normal 20.8% +/- 2.4). All patients had normal myocardial T2* values indicating no myocardial iron deposition. Additional details are provided in Table. Among those who underwent HCT, all 4 had elevated ECV at baseline, which improved in 1 patient 1-month post-HCT (27% [pre] to 21% [post]). One patient had a dilated LA at baseline with the other 3 developing LA dilation at 1-month post HCT. Conclusion CMR provides accurate and reproducible measurements of chamber sizes/ventricular volumes, myocardial mass, and ventricular function. Multiparametric mapping enabled quantitative evaluation of myocardial fibrosis in patients with SCD. We found that diffuse myocardial fibrosis is often present in children with SCD, even those with apparently normal cardiac function. This indicates damage to the myocardium occurs before symptoms or measurable changes on echocardiography, calling into question our current screening procedures. T1 mapping to monitor the progression of fibrosis could help evaluate response to current therapies including SCD directed therapies, cardioprotective medications, and curative options such as HCT and gene therapy. Hence, we believe that cardiac MRI is complementary to echocardiography and provides additional clinically relevant information that will help guide treatment in patients with SCD. Additionally, it has been shown that patients with SCD who undergo successful HCT exhibit significant improvements in cardiac size, function, and diastolic filling parameters as early as 3 months after the procedure, with improvements continuing slowly up to 1 year after HCT. Early post-HCT evaluation in our cohort did show moderate improvement in ECV in one HCT recipient. Further follow up with serial monitoring is ongoing to evaluate long term changes in these patients. Data on additional patients who have undergone serial CMR monitoring after HCT will be presented at the ASH annual meeting. Figure 1 Figure 1. Disclosures Sharma: CRISPR Therapeutics: Other, Research Funding; Medexus Inc: Consultancy; Spotlight Therapeutics: Consultancy; Novartis: Other: Salary support paid to institution; Vertex Pharmaceuticals/CRISPR Therapeutics: Other: Salary support paid to institution; Vindico Medical Education: Honoraria. Hankins: UpToDate: Consultancy; Global Blood Therapeutics: Consultancy; Vindico Medical Education: Consultancy; Bluebird Bio: Consultancy. Triplett: Miltenyi: Other: Travel, meeting registration.