Individuals with heart failure have significantly reduced exercise capacity, a critical life-limiting symptom for those living with the disease. Heart failure is negatively correlated with decreased heart rate variability, including the loss of heart rate variability in tune with breathing—termed respiratory heart rate variability (RespHRV). We tested the hypothesis that restoration of RespHRV would improve exercise tolerance. Heart failure was induced in adult female sheep using a microembolization technique, and the sheep were divided into two groups: RespHRV paced and monotonically paced. Following a 1-week baseline recording, the sheep underwent 2 weeks of pacing. Direct recordings of hemodynamic parameters, including arterial pressure, cardiac output, coronary artery blood flow, and heart rate, were taken at rest and during treadmill exercise. Reinstating RespHRV significantly increased resting cardiac output, a change not observed in monotonically paced sheep. Neither group showed a change in resting coronary artery blood flow. During exercise, RespHRV-paced sheep showed increased cardiac output, coronary artery blood flow, cardiac power output, and faster heart rate recovery post-exercise. In contrast, monotonically paced sheep showed no changes in exercise-induced cardiac function. A separate group of heart failure animals were studied to determine if these benefits would persist alongside heart failure medications. RespHRV pacing continued to improve resting cardiac output with concurrent heart failure medications. Our results indicate that reinstating RespHRV may be a novel approach for improving outcomes in heart failure, including exercise capacity.
A major obstacle to progress in heart failure with preserved ejection fraction (HFpEF) is the paucity of clinically relevant animal models. We developed a large, translationally relevant model in Ossabaw minipigs, which are genetically predisposed to the metabolic syndrome (MetS). Pigs were fed a “Western diet” high in calories, fructose, fat, cholesterol, and salt and received 1–2 deoxy-corticosterone acetate (DOCA) depots (n = 10). After 6 months, they exhibited liver function abnormalities and marked increases in body weight, arterial blood pressure, serum cholesterol and triglycerides, and plasma glucose and insulin levels (glucose tolerance test), indicating the development of a full MetS. Echocardiography demonstrated no change in LV ejection fraction but progressive concentric LV hypertrophy and left atrial dilatation. Doppler echocardiography showed increased E/e’ ratio and increased peak early (E) and peak late atrial (A) transmitral inflow velocities, with no change in E/A ratio. Right heart catheterization demonstrated increased central venous pressure, pulmonary arterial systolic pressure, and pulmonary capillary wedge pressure. Clinically, pigs exhibited impaired exercise capacity, assessed by treadmill tests, associated with chronotropic incompetence. Pathologic examination showed significant myocardial fibrosis, myocyte hypertrophy, and liver fibrosis. In contrast, lean pigs fed a standard diet (n = 3) did not show any changes at 6 months. The Ossabaw porcine model described herein is unique in that it recapitulates the entire constellation of major multiorgan comorbidities and hemodynamic, clinical, and metabolic features of MetS-driven human HFpEF: obesity, arterial hypertension, hyperlipidemia, glucose intolerance, insulin resistance, liver fibrosis and dysfunction, pulmonary hypertension, increased LV filling pressures, concentric LV hypertrophy, LV diastolic dysfunction with preserved systolic function, and impaired exercise capacity. Because of its high clinical relevance, this model is well-suited for exploring the pathophysiology of MetS-driven HFpEF and the efficacy of new therapies.
The plasma membrane ATP-sensitive potassium (KATP) channel in cardiac myocytes plays a critical role in protecting the heart against ischemic injury. Post-translational modifications regulate KATP channel activity and play a role in cardioprotection. However, the role of tyrosine phosphorylation in KATP channel regulation remains unclear. In this study, we investigated the cardiac KATP channel subtype Kir6.2/SUR2A and demonstrated that a protein tyrosine kinase inhibitor significantly increased the current amplitude through blunting the ATP sensitivity of KATP channels without altering the single-channel current or the channel surface expression. Mutation screening identified Y258 in the Kir6.2 subunit as the tyrosine phosphorylation site of the KATP channel. In cardiomyocytes, KATP channel currents can be reversibly enhanced or weakened by inhibiting the tyrosine kinase epidermal growth factor receptor or the protein tyrosine phosphatase 1B. Furthermore, in a perfused mouse heart model, the inhibitor of epidermal growth factor receptor exhibited a significant cardioprotective effect in a KATP channel dependent manner, indicating the pharmacological potential for treatment of ischemic heart disease.
Following myocardial infarction (MI), patients with type 2 diabetes mellitus (T2DM) have poorer prognosis which may be linked to increased susceptibility of coronary microvessels to injury. Interleukin-36 (IL-36) may mediate this injury but its role in the microcirculation of the chronically hyperglycaemic injured heart is unknown. Intravital and laser speckle imaging of the anaesthetised mouse beating heart evaluated the impact of a 16-week high fat diet (HFD)-induced hyperglycaemia ± myocardial ischaemia–reperfusion injury (IR) injury on coronary microvessels. Neutrophil/platelet recruitment, neutrophil extracellular trap formation, cellular necrosis, vascular leakage, vascular tonal changes, functional capillary density, overall ventricular perfusion and levels of circulating inflammatory cytokines were assessed alongside the vasculoprotective ability of an IL-36 receptor antagonist (IL-36Ra). Whether heightened microvessel damage in injured HFD mice was permanent or reversible was investigated after normalising hyperglycaemia through diet reversal (DR). Microcirculatory events assessed were perturbed basally in HFD mice and further after injury. IL-36Ra mitigated these effects and improved infarct size. DR was also beneficial, decreasing neutrophil recruitment to levels below those seen in untreated mice. Mechanistically, benefits of both IL-36Ra and DR could be explained by decreased endothelial oxidative stress and VCAM-1 expression and possibly by raised levels of IL-4/IL-13. Basal changes in chronically hyperglycaemic coronary microvessels that are heightened in the aftermath of reperfusion may explain the poorer outcomes in MI patients with T2DM. These findings are the first to highlight the specific benefits of IL-36 inhibition and reversing hyperglycaemia through dietary modification on the coronary microcirculation in a preclinical model of T2DM.
Myocardial infarction (MI) is a leading cause of heart failure, with thyroid hormone (TH) signaling playing a key role in heart function and postinfarct recovery. Despite evidence of TH administration's safety in cardiac patients, inconsistent therapeutic outcomes and limited understanding of its mechanisms hinder clinical translation. This study aims to investigate the long-term effect of acute triiodothyronine (T3) administration following MI and to elucidate the mechanisms of its cardioprotective actions. To this end, two doses (40 μg/kg) of T3 were administered immediately after injury and 24 h later in a cryoinjury mouse model of left ventricle (LV) infarction. Remarkably T3 administration significantly reduced scar expansion. Echocardiographic analysis conducted 28 days post-injury revealed that T3 administration improved LV remodeling and prevented LV hypertrophy. At molecular level, T3 administration strongly reduced apoptosis in the peri-infarcted area, without inducing cardiac cell proliferation. Furthermore, T3 prevented the accumulation of long-chain acylcarnitines and the subsequent mitochondrial damage. These findings demonstrate that acute T3 treatment following MI improves long-term LV function and reduces LV remodeling by limiting apoptosis in the peri-infarct region and by preserving mitochondrial function and structural integrity.
Cardiogenic shock (CS) is characterized by reduced cardiac output (CO), reduced end-organ perfusion, and high mortality. Medical therapies have failed to improve survival. The ketone body 3-hydroxybutyrate (3-OHB) enhances cardiac function in heart failure and CS. We aimed to elucidate the cardiovascular and cardiometabolic effects of 3-OHB treatment during CS. In a randomized, assessor-blinded crossover design, we studied 16 female pigs (60 kg, 5 months of age). CS was induced by left main coronary artery microsphere injections. Predefined criteria for CS were a 30% reduction in CO or mixed venous saturation (SvO2). Intravenous 3-OHB infusion and a matching control solution were administered for 120 min in random order. Hemodynamic measurements were obtained by pulmonary artery catheterization and a left ventricular (LV) pressure–volume catheter. Myocardial mitochondrial function was assessed using high resolution respirometry. During CS, infusion with 3-OHB increased CO by 0.9 L/min (95%CI 0.4–1.3 L/min) compared with control infusion. SvO2 (P = 0.026) and heart rate (P < 0.001) increased. Stroke volume (P = 0.6) was not altered. LV contractile function as determined by LV end-systolic elastance improved during 3-OHB infusion compared with control infusion (P = 0.004). Systemic and pulmonary vascular resistance decreased, and diuresis increased. LV mitochondrial function was higher after 3-OHB infusion compared with control. We conclude that 3-OHB infusion enhances cardiac function by increasing contractility and reducing vascular resistance, while also preserving myocardial mitochondrial respiratory function in a large animal model of ischemic CS. These novel findings support the therapeutic potential of exogenous ketone supplementation in CS management.
The unsuccessful translation of cardiac regeneration and cardioprotection from animal experiments to clinical applications in humans has raised the question of whether microRNA bioinformatics can narrow the gap between animal and human research outputs. We reviewed the literature for the period between 2000 and 2024 and found 178 microRNAs involved in cardioprotection and cardiac regeneration. On analyzing the orthologs and annotations, as well as downstream regulation, we observed species-specific differences in the diverse regulation of the microRNAs and related genes and transcriptomes, the influence of the experimental setting on the microRNA-guided biological responses, and database-specific bioinformatics results. We concluded that, in addition to reducing the number of in vivo experiments, following the 3R animal experiment rules, the bioinformatics approach allows the prediction of several currently unknown interactions between pathways, coding and non-coding genes, proteins, and downstream regulatory elements. However, a comprehensive analysis of the miRNA-mRNA-protein networks needs a profound bioinformatics and mathematical education and training to appropriately design an experimental study, select the right bioinformatics tool with programming language skills and understand and display the bioinformatics output of the results to translate the research data into clinical practice. In addition, using in-silico approaches, a risk of deviating from the in vivo processes exists, with adverse consequences on the translational research.
Intravenous thrombolysis remains the cornerstone for restoring cerebral reperfusion post-stroke. Despite recombinant tissue plasminogen activator (rtPA) achieving arterial reperfusion within 6 h, persistent microcirculatory blood flow reduction often hampers recovery. Exendin-4, a glucagon-like peptide-1 receptor agonist (GLP-1RA), has demonstrated potential for improving stroke outcomes, though its mechanisms remain partially unclear. This study investigated the role of Exendin-4 in restoring microcirculatory blood flow post-stroke. Using ischemic stroke models in 8-week-old male C57BL/6j mice, induced by transient middle cerebral artery occlusion or bilateral common carotid artery ligation, Exendin-4 (150 μg/kg) was administered intravenously. Infarct size and neurological deficits were evaluated using TTC staining and neurological severity scores. Real-time cerebral blood flow (CBF) and microvascular changes were measured with laser speckle imaging and two-photon microscopy. Mechanistic studies employed immunofluorescence and infrared differential interference contrast microscopy. Our findings demonstrated that Exendin-4 significantly reduced infarct size and improved neurological outcomes, independent of blood glucose levels. Immunofluorescence revealed GLP-1 receptor expression in arteriolar smooth muscle cells, endothelial cells, and pericytes. Exendin-4 enhanced microvascular blood flow via vasodilation, confirmed through real-time imaging. In vitro, Exendin-4 relaxed pre-constricted vessels, an effect that was abolished by eNOS and adenylate cyclase (AC) inhibitors. However, guanylate cyclase (GC) inhibition failed to block Exendin-4-induced vasodilation, suggesting a non-cGMP-dependent NO pathway may be involved. Furthermore, prostaglandin E2 (PGE2) signaling via EP4 receptors was identified as a critical contributor to Exendin-4’s vasodilatory effect, highlighting the involvement of multiple signaling pathways. These findings suggest that Exendin-4 preserves cerebral microcirculation through a multifaceted mechanism involving GLP-1R-mediated AC–cAMP signaling, PGE2–EP4 signaling, and a non-cGMP-dependent NO pathway. This study positions GLP-1 receptor agonists as promising therapeutic candidates for enhancing cerebral microcirculation and improving outcomes following stroke.
Since cell dying in heart failure (HF) may vary based on the aetiology, we examined the main forms of regulated necrosis, such as necroptosis and pyroptosis, in the hearts damaged due to myocardial infarction (MI) or pressure overload. We also investigated the effects of a drug inhibiting RIP3, a proposed convergent point for both these necrosis-like cell death modes. In rat hearts, left ventricular function, remodelling, pro-cell death, and pro-inflammatory events were investigated, and the pharmacodynamic action of RIP3 inhibitor (GSK'872) was assessed. Regardless of the HF aetiology, the heart cells were dying due to necroptosis, albeit the upstream signals may be different. Pyroptosis was observed only in post-MI HF. The dysregulated miRNAs in post-MI hearts were accompanied by higher levels of a predicted target, HMGB1, its receptors (TLRs), as well as the exacerbation of inflammation likely originating from macrophages. The RIP3 inhibitor suppressed necroptosis, unlike pyroptosis, normalised the dysregulated miRNAs and tended to decrease collagen content and affect macrophage infiltration without affecting cardiac function or structure. The drug also mitigated the local heart inflammation and normalised the higher circulating HMGB1 in rats with post-MI HF. Elevated serum levels of HMGB1 were also detected in HF patients and positively correlated with C-reactive protein, highlighting pro-inflammatory axis. In conclusion, in MI-, but not pressure overload-induced HF, both necroptosis and pyroptosis operate and might underlie HF pathogenesis. The RIP3-targeting pharmacological intervention might protect the heart by preventing pro-death and pro-inflammatory mechanisms, however, additional strategies targeting multiple pro-death pathways may exhibit greater cardioprotection.
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