Citations

Background: Heart failure with preserved ejection fraction (HFpEF) accounts for approximately half of all heart failure hospitalizations; yet, tools for individualized 1-year mortality risk stratification remain limited. We conducted a retrospective study to develop and validate a machine learning model using genetic programming and automated model search to predict 1-year all-cause mortality in HFpEF patients admitted for decompensated HF. Methods: Electronic medical records from Mayo Clinic (2010-2020) were queried to identify 7,840 adult patients hospitalized with HF exacerbation and a left ventricular ejection fraction>50% on echocardiograms performed between six months pre-admission and seven days post-discharge. Demographic, clinical, laboratory, medication, comorbidity, and imaging data available at the time of index hospitalization were extracted. The cohort was split into 80% training (n=6,272) and 20% held-out test (n=1,568) sets. After standard preprocessing, we performed an evolutionary AutoML search. The final pipeline applied a RobustScaler, two sequential feature-union blocks, and an XGBoost classifier. Model performance on the test set was assessed by area under the receiver operating characteristic curve (AUC), Brier score, accuracy, precision, recall, and F1 score, each with bootstrap-derived 95% confidence intervals (CIs). For interpretability, SHAP (SHapley Additive exPlanations) values were computed using the full scaled training set as background. Results: On the held-out test set, the XGBoost-based model achieved an AUC of 0.7595 (95% CI: 0.7307-0.7870) and a Brier score of 0.1764 (95% CI: 0.1652-0.1890). Accuracy was 0.7406 (95% CI: 0.7143-0.7645), precision 0.6349 (95% CI: 0.5759-0.6942), recall 0.4135 (95% CI: 0.3636-0.4609), and F1 score 0.5008 (95% CI: 0.4529-0.5444). The global SHAP summary plot (Figure 1) ranked age, serum albumin, blood urea nitrogen (BUN), presence of renal failure, and NT-proBNP as the top 5 predictors. Conclusions: In this large HFpEF cohort, an AutoML-derived XGBoost pipeline achieved robust discrimination (AUC≈0.76) and calibration (Brier≈0.18) for 1-year mortality prediction. SHAP-based explanations highlighted the importance of age, nutritional status (albumin), renal biomarkers (BUN, renal failure), and natriuretic peptides (NT-proBNP) as principal drivers of risk. These interpretable machine learning findings may guide personalized risk stratification and identify therapeutic targets in HFpEF.

Abstract Background One in five adults aged 40 will develop heart failure (HF) during their lifetime. Risk factors (e.g., hypertension, diabetes mellitus, coronary artery disease, etc.) lead to structural and functional changes in cardiomyocytes, resulting in HF. At the cellular level, these changes consist of mis/over‐expression of genes that regulate cardiac identity (e.g., CamK2δ, PKC, Stat3, etc.). The current paradigm for treating HF is pharmacological or device‐based intervention; however, with few exceptions, the condition worsens with time. We are proposing to implement a change in HF treatment, shifting from a drug‐centric system to a cardiac target‐specific molecular approach that would reverse hypertrophy and adverse remodelling of affected cardiomyocytes. Methods A cardiomyocyte targeting peptide (CTP) was reversibly linked to miRNA106a for delivery to a mouse model of HF. Reversal of morphological, signalling, and physiological HF parameters was measured. Additionally, CTP‐miRNA106a was introduced into a human cardiomyocyte cell line to identify mechanism(s) at play for reversing HF characteristics (e.g., hypertrophy). Results Bio‐distribution studies showed that intravenously injected CTP‐miRNA106 delivered its cargo specifically to the heart within 30 min, followed by clearance of CTP from the heart to the kidneys, and to a lesser extent, the liver by 35 h with persistence of miRNA106a in cardiomyocytes until day 7 (the latest tested time‐point). CTP‐miRNA106a reversed angiotensin2/isoproterenol‐induced hypertrophy in 90% of the treated mice. We also identified two potential HF intracellular signalling pathways/mechanisms (PLCβ1/PKC/IP3 and NF‐κB) targeted by CTP‐miRNA106a that could benefit many pathophysiologies underlying HF, including inflammation. Conclusions CTP‐miRNA106a, a first‐of‐a‐kind cardiac‐specific drug, downregulates genes involved in cardiac hypertrophy and inflammation through the PLCβ1 and CamKIIδ kinase pathways. CTP delivery of miRNA106a cargo is specific to cardiomyocytes both in vitro and in vivo, and once delivered, many HF parameters, including hypertrophy, are reversed. Key points Cardiac Targeting Peptide (CTP) delivers, specifically to the heart, reversibly linked miRNA106a. MiRNA106a targets genes in various pathways known to cause heart failure when they are over/hyper activated returning their expression to normal levels. In vivo analyses using a mouse heart failure model resulted in reversal of heart failure parameters in 19/20 mice.

Introduction: Heart failure with preserved ejection fraction (HFpEF) is an increasingly prevalent form of heart failure (HF) in the US today. Our prior work revealed low albumin at first hospitalization for HF exacerbation with underlying HFpEF to be an independent predictor of all-cause mortality in a small cohort of patients. We now sought to confirm our earlier findings across a larger and more diverse patient population. Methods: Seven thousand, eight hundred and forty patients had a first admission to Mayo Clinic for HF exacerbation with an echo-confirmed left ventricular ejection fraction>50% between 2010 and 2020. Patient baseline demographics, co-morbidities, admission laboratory values, echocardiographic parameters, discharge medications, and outcomes were obtained from chart abstraction. To validate our previous model, patients were grouped based on the number of risk factors as previously defined: age>80 years, serum albumin level<3.2 g/dl, medial E/e'≥20, N-terminal pro-brain natriuretic peptide (NT-proBNP) >5,000 pg/mL, and the presence of hypertension (HTN). The association with 1 year mortality was studied using Cox regression. Kaplan Meier curves were generated based on the number of factors present per patient. Results: Eleven significant univariate predictors of all-cause mortality were identified from the data (Fig. 1A), with hazard ratios (HR) ranging from 1.22-2.04 (p<0.001 for all). Notably, four previously identified prognosticators (age, albumin, medial E/e’, , HTN) were noted as significant independent predictors of all-cause mortality in HFpEF (HR 1.67, 2.14, 1.18, and 1.15, respectively, p<0.004 for all ; Fig. 1B). Multivariate regression modeling identified low albumin on presentation as being a significant adverse prognosticator (HR=2.14; p-value<0.001). Conclusion: Our analysis of 7840 HFpEF patients confirms our prior findings, suggesting that this model is a strong predictive tool for assessing the prognosis of HFpEF. Taken together, these five variables are easily available in the in-patient setting and can be incorporated into a model predictive of 1-year all-cause mortality.

Introduction: Intracellular calcium handling plays a central role in cardiac contractility and relaxation. Genes such as the Ca 2+ -ATPase pump (SERCA2a) and the Dihydropteridine reductase (DHPR) enzyme are especially important in the cycling of intracellular calcium. As prior research has established a link between treatment with our cardiac targeting peptide (CTP: a 12-amino acid, cardiomyocyte-specific cell penetrating peptide) and upregulation of calcium-handling genes such as SERCA2a and DHPR, we hypothesized that treatment with our novel hybrid cardiac targeting peptide (hCTP: positions #3 and 11 replaced with the D-enantiomer form of the amino acids) will improve intracellular calcium handling in human cardiomyocytes (CMCs) by increasing expression of key calcium handling genes instrumental in sequestering calcium in diastole and lowering cytosolic levels. Methods: To test hCTP’s calcium handling abilities, CMCs were plated on day 1 and allowed to settle for 24 hours, after which they received daily treatment with various concentrations of hCTP (1-25µM) for three days. On day 5, cells underwent either intracellular calcium measurements using the FLIPR 6 Calcium assay or were prepared for western blotting. Western blotting was performed to determine the protein concentrations of SERCA2a and DHPR, as compared to a GAPDH control. To test hCTP’s intrinsic calcium handling abilities, various concentrations of hCTP were mixed with a constant amount of elemental calcium dissolved in water. After 20 min at 37 o C, the concentration of free Ca 2+ was measured with an ABL90 Flex Plus radiometer. Results: In CMCs, treatment with hCTP led to a decrease in cytosolic calcium in a dose dependent fashion; concentrations as low as 1µM led to a significant decrease in cytosolic calcium (p< 0.0001 for all groups). Western blots performed on the treated CMCs revealed an increase in expression of both DHPR and SERCA2a, normalized to GAPDH control. Outside of the cellular environment, hCTP did not appear to display inherent calcium sequestring properties, suggesting that improvements in calcium handling are due to upregulation of gene expression. Conclusion: hCTP appears to lower cytosolic calcium and improve calcium handling through upregulation of SERCA2a and DHPR. Our data indicates that hCTP is worth pursuing as a stand-alone, cardio-selective therapy for cardiomyopathies involving calcium mishandling.

Introduction: Role of NF-κB driven inflammation leading to heart failure with preserved ejection fraction has received little attention. Our previous in vivo work with cardiac targeting peptide (CTP) in guinea pigs identified suppression of NF- κB pathway by CTP. In the current work we sought to further study the effects of a hybrid version of CTP (hCTP: amino acids at positions 3 and 11 substituted with D-amino acids) on TNF-α mediated NF-κB activation, as well as expression of its downstream inflammatory markers IL-1β and IL-8. Methods: Human cardiomyocytes (CMCs) were transfected using Lipofectamine 3000 with a reporter luciferase plasmid carrying an NF-κB promoter site upstream of the luciferase gene. Cells were treated with various concentrations of hCTP for 24 hours, followed by a TNF-α challenge (20ng/mL), addition of luciferin as substrate, and measurement of luminescence. Cell viability in response to above manipulations was assessed by FACS using live-dead stain. CMCs were treated as above, and cell culture supernatants analyzed for IL-8 levels using human ELISA kit. In another set of experiments, CMCs were transfected with NF-κB reporter plasmid, and after treatment for 24 hours with various concentrations of hCTP, incubated with 10nM angiotensin 2 (Ang2) and 100nM phenylephrine (PE) for 24hrs to assess levels of IL-1β in the cell culture media using an ELISA assay. Results: hCTP inhibited TNF-α mediated NF-κB activation with a dose-response curve, with maximum inhibition with hCTP seen at 10µM concentrations (p<0.001). Treatment with hCTP inhibited the TNF-α induced IL-8 secretion with significant decreases seen even at the 1µM dose. Similarly, secretion of IL-1β production by CMCs in response to Ang2/PE stimulation was suppressed by hCTP even at the lowest tested dose (1µM; p<0.5). Conclusions: Our data suggests that hCTP inhibited NF-κB pathway transcriptional activity in response to TNF-α stimulation in a dose-dependent fashion in human CMCs. Furthermore, treatment with hCTP inhibited TNF-α induced IL-8 and IL-1β secretion even at doses as low as 1µM. The ability of hCTP to efficiently targeting this inflammatory pathway suggests that it has therapeutic potential in the treatment of heart failure. Further in vivo studies are ongoing to test this hypothesis.

Introduction: Heart failure with preserved ejection fraction (HFpEF) is a form of heart failure which is rapidly rising in incidence in the US today. Despite this, no therapies with mortality benefit exist for this syndrome. Previous work with our novel hybrid cardiac targeting peptide (hCTP), a cardiomyocyte-specific cell penetrating peptide, has shown multiple beneficial biological effects (improved calcium handling, anti-inflammatory properties) beyond its transduction abilities. Therefore, we hypothesized that treatment with hCTP in a mouse model of HFpEF would ameliorate the pathophysiology of this disease. Methods: To establish a HFpEF model, 6-week-old, C57BL/6NJ mice were placed on a high-fat diet (HFD), 60% kCal from lard, and water with 0.5 g/L of an iNOS inhibitor (N-Nitro-L-arginine methylester) to induce hypertension. After 5 weeks of the diet, HFD mice and their littermate controls underwent baseline exercise testing and echocardiograms. Exercise capacity was measured after 2 days of exercise training with 2 days of rest in between; mice were placed on a treadmill at a 10-degree angle and made to run at increasingly fast speeds (5-30 m/min) until exhaustion. Echocardiograms were performed under 1.5% inhalational isoflurane. After baseline testing, the mice were treated with vehicle, or either 2.5 or 5 mg/Kg hCTP, either 3 or 5 days a week (five groups, n = 3 per group). After four weeks of treatment, the mice were again subjected to the same battery of tests and euthanized. Data was compared using two way ANOVA with Sidak’s test to compare measurements pre- and post-treatment. Results: Treatment with hCTP at either concentration for five days/week led to an increase in running distance from 39.8 m to 116.3 m (p = 0.03, n = 6), compared to changes in control distance from 208.4 m to 157.8 m (p = 0.399). Running distance did not correlate with weight (R 2 < .001). Treatment with 5 mg/Kg hCTP for 5 days/week led to a normalization of the E/e’ ratio, from 43.3 to 26.9 (p = 0.004, n = 3) compared to a change in the control of 34.7 to 26.1 (p = 0.312). Ejection fractions remained consistent (>55%) across all groups. Conclusions: Our data suggests that hCTP appears to be a promising treatment in ameliorating some of the physiological effects in a murine model of HFpEF. Gene and protein expression studies in heart tissue are underway. Further work is warranted with a larger sample size.

One of the bottlenecks to bringing new therapies to the clinic has been a lack of vectors for delivering novel therapeutics in a targeted manner. Cell penetrating peptides (CPPs) have received a lot of attention and have been the subject of numerous developments since their identification nearly three decades ago. Known for their transduction abilities, they have generally been considered inert vectors. In this review, we present a schema for their classification, highlight what is known about their mechanism of transduction, and outline the existing literature as well as our own experience, vis a vis the intrinsic anti-inflammatory properties that certain CPPs exhibit. Given the inflammatory responses associated with viral vectors, CPPs represent a viable alternative to such vectors; furthermore, the anti-inflammatory properties of CPPs, mostly through inhibition of the NF-κB pathway, are encouraging. Much more work in relevant animal models, toxicity studies in large animal models, and ultimately human trials are needed before their potential is fully realized.

Targeted delivery of therapeutics specifically to cardiomyocytes would open up new frontiers for common conditions like heart failure. Our prior work using a phage display methodology identified a 12-amino-acid-long peptide that selectively targets cardiomyocytes after an intravenous injection in as little as 5 min and was hence termed a cardiac-targeting peptide (CTP: APWHLSSQYSRT). CTP has been used to deliver imaging agents, small drug molecules, photosensitizing nanoparticles, exosomes, and even miRNA to cardiomyocytes. As a natural extension to the development of CTP as a clinically viable cardiac vector, we now present toxicity studies performed with the peptide. In vitro viability studies were performed in a human left ventricular myocyte cell line with 10 µM of Cyanine-5.5-labeled CTP (CTP-Cy5.5). In vitro ion channel profiles were completed for CTP followed by extensive studies in stably transfected cell lines for several GPCR-coupled receptors. Positive data for GPCR-coupled receptors were interrogated further with RT-qPCRs performed on mouse heart tissue. In vivo studies consisted of pre- and post-blood pressure monitoring acutely after a single CTP (10 mg/Kg) injection. Further in vivo toxicity studies consisted of injecting CTP (150 µg/Kg) in 60, 6-week-old, wild-type CD1, male/female mice (1:1), with cohorts of mice euthanized on days 0, 1, 2, 7, and 14 with inhalational CO2, followed by blood collection via cardiac puncture, complete blood count analysis, metabolic profiling, and finally, liver, renal, and thyroid studies. Lastly, mouse cardiac MRI was performed immediately before and after CTP (150 µg/Kg) injection to assess changes in cardiac size or function. Human left ventricular cardiomyocytes showed no decrease in viability after a 30 min incubation with CTP-Cy5.5. No significant activation or inhibition of any of seventy-eight protein channels was observed other than OPRM1 and COX2 at the highest tested concentration, neither of which were expressed in mouse heart tissue as assessed using RT-qPCR. CTP (10 mg/Kg) injections led to no change in blood pressure. Blood counts and chemistries showed no evidence of significant hematological, hepatic, or renal toxicities. Lastly, there was no difference in cardiac function, size, or mass acutely in response to CTP injections. Our studies with CTP showed no activation or inhibition of GPCR-associated receptors in vitro. We found no signals indicative of toxicity in vivo. Most importantly, cardiac functions remained unchanged acutely in response to CTP uptake. Further studies using good laboratory practices are needed with prolonged, chronic administration of CTP conjugated to a specific cargo of choice before human studies can be contemplated.

Background: Amiodarone is underutilized due to significant off-target toxicities. We hypothesized that targeted delivery to the heart would lead to the lowering of the dose by utilizing a cardiomyocyte-targeting peptide (CTP), a cell-penetrating peptide identified by our prior phage display work. Methods: CTP was synthesized thiolated at the N-terminus, conjugated to amiodarone via Schiff base chemistry, HPLC purified, and confirmed with MALDI/TOF. The stability of the conjugate was assessed using serial HPLCs. Guinea pigs (GP) were injected intraperitoneally daily with vehicle (7 days), amiodarone (7 days; 80 mg/kg), CTP–amiodarone (5 days; 26.3 mg/kg), or CTP (5 days; 17.8 mg/kg), after which the GPs were euthanized, and the hearts were excised and perfused on a Langendorff apparatus with Tyrode’s solution and blebbistatin (5 µM) to minimize the contractions. Voltage (RH237) and Ca2+-indicator dye (Rhod-2/AM) were injected, and fluorescence from the epicardium split and was captured by two cameras at 570–595 nm for the cytosolic Ca2+ and 610–750 nm wavelengths for the voltage. Subsequently, the hearts were paced at 250 ms with programmed stimulation to measure the changes in the conduction velocities (CV), action potential duration (APD), and Ca2+ transient durations at 90% recovery (CaTD90). mRNA was extracted from all hearts, and RNA sequencing was performed with results compared to the control hearts. Results: The CTP–amiodarone remained stable for up to 21 days at 37 °C. At ~1/15th of the dose of amiodarone, the CTP–amiodarone decreased the CV in hearts significantly compared to the control GPs (0.92 ± 0.05 vs. 1.00 ± 0.03 ms, p = 0.0007), equivalent to amiodarone alone (0.87 ± 0.08 ms, p = 0.0003). Amiodarone increased the APD (192 ± 5 ms vs. 175 ± 8 ms for vehicle, p = 0.0025), while CTP–amiodarone decreased it significantly (157 ± 16 ms, p = 0.0136), similar to CTP alone (155 ± 13 ms, p = 0.0039). Both amiodarone and CTP–amiodarone significantly decreased the calcium transients compared to the controls. CTP–amiodarone and CTP decreased the CaTD90 to an extent greater than amiodarone alone (p < 0.001). RNA-seq showed that CTP alone increased the expression of DHPR and SERCA2a, while it decreased the expression of the proinflammatory genes, NF-kappa B, TNF-α, IL-1β, and IL-6. Conclusions: Our data suggest that CTP can deliver amiodarone to cardiomyocytes at ~1/15th the total molar dose of the amiodarone needed to produce a comparable slowing of CVs. The ability of CTP to decrease the AP durations and CaTD90 may be related to its increase in the expression of Ca-handling genes, which merits further study.