Effects of intravenous furosemide plus small-volume hypertonic saline solutions on inflammatory, remodelling markers and epigenetics signatures of patients with congestive acute decompensated heart failure (ADHF)

Introduction

Heart failure (HF) is one of the principal causes of mortality in industrialized countries, particularly in ageing populations. Advances in coronary revascularization techniques have contributed to a decline in deaths from acute myocardial infarction; however, they have also led to a progressive rise in the prevalence of post-ischemic HF. In contrast to the substantial progress in revascularization strategies, genuinely innovative clinical approaches aimed at targeting the wound-healing phase of myocardial injury and favorably modulating cardiac remodeling remain relatively limited [1, 2].

HF is a complex multisystem syndrome characterized by impaired cardiac performance. A reduction in cardiac output, largely due to left ventricular dysfunction, triggers compensatory activation of neurohormonal pathways. This, in turn, stimulates the renin–angiotensin–aldosterone system, resulting in increased levels of renin, angiotensin II and aldosterone, which promote sodium and water retention, vasoconstriction and heightened sympathetic drive. Chronic exposure to this neurohormonal activation induces ventricular dilatation, structural myocardial remodeling and fibrosis, thereby further compromising cardiac output [3, 4].

The HF syndrome encompasses both microstructural and macrostructural alterations, each accompanied by activation of inflammatory and neurohormonal cascades that release a wide array of biomolecules in an attempt to compensate for the failing heart. As a consequence, a “storm” of cytokines and regulatory mediators is generated. The sheer number and dysregulation of these molecules make it challenging to identify specific biomarkers that are closely linked to the clinical setting of acute decompensated heart failure (ADHF).

These dysregulated molecules may serve in clinical and scientific practice as potential remodeling markers such as suppression of tumorigenicity 2 (ST2), Galectin-3mm, troponin-T, or potential cardiac stretching markers such as NT-proBNP, or inflammatory markers such as some proinflammatory cytokines. Furthermore, the epigenetic alterations such as the microRNAs expression changes occurring in the clinical context of the heart failure syndrome may be used as epigenetic signatures of heart failing.

In response to mechanical strain, cardiomyocytes synthesize suppression of tumorigenicity 2 (ST2), which is present in two isoforms: a transmembrane/cellular form (ST2L) and a soluble circulating form (sST2) [5]. ST2 functions as the receptor for interleukin-33 (IL-33), a cytokine released following cellular injury and predominantly expressed by endothelial and epithelial cells [6]. Elevated levels of sST2 act as a decoy receptor, sequestering IL-33 and thereby limiting its interaction with ST2L; this mechanism attenuates the cardioprotective effects of IL-33 observed in experimental models, including the reduction of myocardial fibrosis, cardiomyocyte hypertrophy and apoptosis [7].

Galectin-3, predominantly produced by macrophages but also expressed by other cell types, promotes fibroblast proliferation and stimulates collagen deposition within the myocardium [8, 9]. Several studies have demonstrated that increased circulating Galectin-3 levels are associated with newly diagnosed HF and that this biomarker provides additional value as an indicator of prognosis and disease severity in patients with both HFrEF and HFpEF [10, 11].

Troponin is a regulatory protein complex of the sarcomere, composed of three distinct subunits (I, C, and T). Elevated serum troponin concentrations correlate with greater disease severity and higher mortality risk, whereas decreasing levels are associated with a more favorable prognosis [12].

Natriuretic peptides (NPs) are synthesized in the heart in response to atrial and ventricular wall stretch and neurohormonal activation [13]. Both BNP (B-type natriuretic peptide) and NT-proBNP (N-terminal pro-B-type natriuretic peptide) are routinely employed to support diagnosis, monitor therapeutic efficacy, and estimate prognosis in patients with HFrEF [14].

Proinflammatory cytokines, including interleukin-6 (IL-6), interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-α), are upregulated in individuals with HF, and their circulating levels show a direct relationship with both New York Heart Association (NYHA) functional class and left ventricular ejection fraction (EF) [15–17].

Circulating microRNA (miRNA) levels change dynamically in the setting of multiple acute and chronic conditions, and their remarkable stability in stored biological samples supports their potential as biomarkers in HF [18]. However, a clear agreement is still lacking regarding which specific serum miRNAs might represent the optimal markers for HF. Emerging data indicate that distinct miRNAs are differentially regulated in the failing myocardium, influencing several dimensions of the HF phenotype [19, 20, 21]. MiRNAs have been implicated in HF pathogenesis by negatively modulating the expression of genes that orchestrate adaptive and maladaptive cardiac remodeling. Prior investigations have documented profound changes in myocardial miRNA expression profiles in HF, while other studies have demonstrated their involvement in key signaling pathways implicated in disease progression [19, 22].

In a notable study, investigators explored whether left ventricular reverse remodeling after cardiac resynchronization therapy (CRT) in patients with HF and ventricular dyssynchrony was linked to modifications in circulating miRNAs [23]. They observed that responders exhibited higher expression of five miRNAs implicated in cardioprotection against adverse remodeling. The authors concluded that a favorable therapeutic response in terms of reverse remodeling is accompanied by beneficial shifts in miRNAs that regulate cardiac fibrosis, apoptosis and hypertrophy.

In both experimental models and human myocardium, it is generally accepted that alterations in cardiomyocyte biology represent the principal trigger for cardiac remodelling, although some experimental settings demonstrate that remodelling can develop even in the absence of overt myocyte dysfunction. Reverse remodelling, or cardiac de-remodelling, denotes the partial or complete reversal of disease- or injury-induced structural and functional changes, particularly in the context of HF. It is characterized by a reduction in chamber dimensions, especially of the left ventricle, together with improved contractile performance. This phenomenon is usually interpreted as a marker of myocardial recovery and is often a key therapeutic objective in HF management [24]. On the basis of current evidence, several microRNAs have been shown to regulate and/or modulate critical elements of the hypertrophic programme in cardiac myocytes, including reactivation of the so-called fetal gene pattern, and may also participate in processes of reverse remodelling [22, 23].

A number of miRNAs have been linked to pathological pathways involved in HF development and progression [21–23, 25]. Among these, miRNA-365 (miR-365) has emerged as an important member of the miRNA family, with several studies indicating a pivotal role in the evolution of pathological ventricular dysfunction. In an expression-profiling study, Jentzsch et al. [9] observed that miR-365 was markedly upregulated and positively correlated with the degree of myocardial hypertrophy, suggesting a potential pro-hypertrophic action of this miRNA [26].

The miR-125 family, and particularly miR-125a-5p, has also been implicated in HF, especially in the setting of myocardial infarction (MI). Experimental data indicate that miR-125a-5p exerts anti-apoptotic effects in cardiomyocytes, thereby conferring protection against ischemia-induced cell death. Furthermore, one study reported that miR-125a-5p expression patterns could discriminate HFREF from HFPEF [27].

More recently, miR-150-5p has been proposed as a novel circulating biomarker for AHF. Its association with maladaptive remodelling, disease severity and adverse outcomes supports the pathophysiological significance of reduced miR-150-5p expression in AHF [28].

MicroRNA-181b (miR-181b) has likewise been identified as both a potential biomarker and a putative therapeutic target in HF. Altered miR-181b levels have been reported in patients with HF, and accumulating data suggests that it contributes to the regulation of inflammatory responses and cardiac remodelling. A recent study further indicated that miR-181b may serve as a promising circulating biomarker for HF [29].

The circulating expression profile of miR-214 and its contribution to HF pathogenesis has been comparatively less explored. However, a recent investigation clarified the biological and clinical relevance of miR-214 dysregulation in HF. Mechanistically, miR-214 overexpression was shown to impair angiogenesis in HUVECs by targeting XBP1, a key transcription factor in the unfolded protein response; silencing XBP1 reproduced the reductions in HUVEC proliferation and angiogenic capacity observed with miR-214 overexpression. Collectively, these data indicate that miR-214 is a critical modulator of cardiac angiogenesis in vitro and in vivo, most likely through regulation of XBP1 expression, and support a central role for miR-214 in the control/inhibition of angiogenic processes in the failing heart [30].

Preliminary investigations [21, 25] have demonstrated that the use of an intravenous combination of hypertonic saline solution (HSS) and high-dose furosemide is both safe and well tolerated in patients with congestive HF. These studies were based on the hypothesis that preserving adequate intravascular volume and renal perfusion during high-dose loop diuretic therapy may potentiate its diuretic efficacy. Both objectives can be pursued by coupling high-dose furosemide with HSS administration.

In this context, our group previously showed that, in patients with ADHF, treatment with furosemide plus HSS, as compared with furosemide alone, led to a significant reduction in circulating ANP, BNP, TNF-α, IL-1β and IL-6 levels. We further reported that, following an 8-day course of the furosemide + HSS regimen, an acute saline challenge (15 mL/kg of 0.9% NaCl) induced a smaller relative increase in serum ANP, BNP, tumor necrosis factor and IL-1β in these patients compared with control groups [31, 32].

Further studies, more recently, showed the superiority of combination HSS plus furosemide therapy over furosemide alone in terms of kidney function preservation, improved diuresis and natriuresis, weight loss, duration of hospital stay and mortality confirming that in both hospitalized and ambulatory patients with heart failure syndrome, adding HSS to furosemide may improve short-term natriuretic response and outcomes [33–37].

Nevertheless, the possible effects of the i.v. furosemide + HSS treatment on natriuretic and inflammatory markers of heart failure deserve further confirmation, whereas the effects of this type of treatment on epigenetic signatures of pathologic mechanisms involved in the left ventricular dysfunction involved in AHF pathogenesis seem to be still not studied.

Materials and Methods

We prospectively enrolled all consecutive patients older than 18 years admitted to the Internal Medicine Unit of Policlinico “P. Giaccone” in Palermo with a diagnosis of ADHF due to HFrEF between March 2019 and December 2021. Eligible patients were randomly allocated, in a 1:1 ratio, to receive either moderate/high doses of i.v. furosemide in combination with HSS or i.v. furosemide alone, according to a computer-generated randomization list.

Results

We enrolled 216 patients with ADHF and HFrEF admitted to our Internal Medicine ward from March 2019 to December 2021.

Thirteen patients were excluded based on the presence of exclusion criteria. Three patients died during the study period. Thus, 107 patients were randomized to treatment with i.v. high-dose furosemide plus HSS, whereas 93 patients were randomized to i.v. high-dose furosemide alone.

General, demographic and laboratory variables in subjects treated with furosemide plus HSS versus furosemide alone group are listed in Table 1.

Baseline, demographic and laboratory variables in patients randomized to receive i.v. furosemide plus HSS (Group A) and i.v. furosemide alone (Group B) group are reported in Table 1.

Subjects randomized to receive the furosemide + HSS showed at baseline a significantly higher frequency of hypertension (96.2% vs 55.9%; p<0.0005), higher systolic blood pressure (SBP) (135.0±22.04 mm/Hg vs 120.5 ±13.8 mm/Hg; p<0.0005), higher BMI (28.5±5.6 kg/m² vs 25.7±4.5 kg/m²; p<0.0005), higher mean serum total cholesterol (134.2±35.9 mg/dL vs 106.1±74.2 mg/dL; p=0.001), higher mean serum triglycerides (93.3±35.3 vs 64.9±26.4 mg/dL; p<0.0005) in comparison to patients randomized to receive furosemide alone.

Following the treatment phase, the increase in urine output was greater in the furosemide + HSS group than in the furosemide-only group (1021.62±212.29 vs 2340.74±466.37 mL; p<0.0005 and 1004.57±146.70 vs 1907.35±269.36 mL; p<0.0005). In parallel, patients receiving i.v. furosemide + HSS experienced a more pronounced reduction in body weight compared with those treated with i.v. furosemide alone (73.8±5.2 vs 67.5±5.3; p<0.0005 and 72.9±4.1 vs 69.7±4.1; p<0.0005).

Discussion

Our data indicate that, in patients with ADHF and HFrEF, treatment with moderate-to-high doses of i.v. furosemide in combination with HSS, compared with furosemide alone, is associated with a significantly greater reduction in circulating levels of IL-6, sST2, hsTnT, galectin-3 and NT-proBNP.

In a previous study of 94 patients with refractory chronic HF who received i.v. furosemide (500–1,000 mg) plus HSS twice daily, investigators reported a marked increase in daily diuresis and natriuresis, a more rapid decline in BNP concentrations, shorter hospital stays and a lower 30-day readmission rate [38].

In another recent study from our group [39], we observed a comparable magnitude of reduction in three key HF biomarkers, suggesting that the decrease in circulating HF markers is not solely attributable to more pronounced decongestion or faster attainment of clinical compensation. This observation raises the possibility that the therapeutic benefits of this regimen may extend beyond simple relief of congestion-related signs and symptoms. In line with this, the higher delta values documented after treatment with i.v. furosemide plus HSS in the present study point to a potentially greater biological efficacy, possibly mediated through more favourable modulation of myocardial stretch and fibrotic pathways.

Overall, our findings are consistent with prior reports showing superior clinical improvement with i.v. furosemide + HSS compared with furosemide alone, although relatively few studies have specifically evaluated the impact of this strategy on inflammatory and fibrosis-related HF biomarkers [21, 25, 26, 38].

It is well established that a variety of biologically active mediators, including cytokines, are upregulated in the context of HF [36, 37, 40]. In particular, circulating TNF-α and IL-6 levels are elevated and correlate with HF severity [9]. Previous work has demonstrated that optimization of conventional HF therapy with diuretics, ACE inhibitors, beta-blockers and digoxin can significantly reduce serum TNF and IL-6 concentrations [41].

Among the numerous emerging biomarkers, sST2 has been identified as one of the most promising in recent investigations. ST2, a member of the interleukin-1 receptor family also known as interleukin-1 receptor-like 1 (IL1RL1), has been reported to be expressed by cardiac cells in response to myocardial stress [42–45].

Over the past decade, several studies have measured soluble ST2 in different HF settings, generating robust and reproducible evidence for its prognostic value, both additive to and independent of established markers [46–48]. Alternative splicing of the ST2 gene gives rise to multiple isoforms, including a transmembrane receptor (ST2L) and a soluble circulating form (sST2, often simply referred to as ST2), which has gained particular relevance as a biomarker. Both ST2L and sST2 are produced by cardiomyocytes and cardiac fibroblasts in response to mechanical stress, and both bind interleukin-33 (IL-33). IL-33 itself is induced by cellular stretch and appears to exert anti-fibrotic and anti-hypertrophic effects in mechanically stressed tissues through activation of MyD88 (myeloid differentiation primary response 88), IRAK (interleukin-1 receptor-associated kinase), ERK (extracellular signal-regulated kinase) and, ultimately, NF-κB (nuclear factor-κB).

Several studies have shown that the combination of furosemide with HSS is more effective than furosemide alone in improving congestion. Achieving adequate systemic decongestion while preserving or improving renal function is a central goal in HF management [34–40, 49]. When quantified by the degree of haemoconcentration, more complete decongestion has been associated with lower all-cause and cardiovascular mortality and reduced rehospitalization, even in the presence of transient worsening renal function [17, 21, 25, 50, 51]. Beyond this, the addition of HSS to i.v. furosemide may have favourable pleiotropic effects on cardiac remodelling and inflammatory pathways that characterize the congestive HF syndrome [52–56].

Some reports have suggested that furosemide combined with HSS can protect renal function, thereby enhancing diuretic response and improving clinical outcomes [51, 57]. However, the overall efficacy of HSS plus furosemide in acute HF remains debated. Other studies have found comparable effects between combined HSS–furosemide therapy and furosemide alone, with no significant differences in serum creatinine, body weight or urine output [58, 59].

Extracellular volume expansion is a fundamental pathophysiological feature of HF. Excess extracellular fluid leads to elevated intracardiac filling pressures, which in turn give rise to the cluster of signs and symptoms collectively referred to as congestion. Loop diuretics remain a mainstay of HF therapy, yet, unlike other HF treatments, high-quality randomized trial evidence to guide their optimal use is limited. Effective and safe diuretic management in both inpatient and outpatient settings requires a sophisticated understanding of renal physiology and diuretic pharmacokinetics. Diuretic resistance—defined as an inadequate natriuretic response despite appropriate diuretic dosing—represents a major therapeutic challenge and is generally associated with an adverse prognosis [60, 61].

A recent meta-analysis reported that HSS combined with furosemide increases 24-hour urine output and reduces body weight and serum creatinine in patients with acute HF. Furthermore, patients receiving HSS plus furosemide had lower rates of rehospitalization and mortality compared with those treated with furosemide alone [60].

In our cohort, we also observed that, after attaining clinical compensation and undergoing an acute volume challenge with a rapid i.v. saline load, both patients treated with high-dose furosemide plus HSS and those receiving furosemide alone experienced increases in serum IL-6, sST2 and galectin-3, but not in NT-proBNP. Notably, the magnitude of the increase in IL-6, sST2 and galectin-3 was significantly smaller in the group treated with i.v. furosemide + HSS than in the furosemide-only group, further supporting a more favourable modulation of inflammatory and remodelling pathways with the combined regimen. Some previous studies sustain the use of an isotonic saline load to challenge clinical compensation in patients with ventricular dysfunction [59–61].

In our previous work [32], we also found that, following an acute saline load, patients treated with high-dose i.v. furosemide plus HSS exhibited a significantly smaller percentage change in natriuretic peptides and immuno-inflammatory markers compared with control groups.

Several studies have examined ANP and BNP responses to acute saline infusion. Langer et al. [62] reported that an i.v. saline bolus (18 mL/kg sodium chloride at 250 mL/min) in healthy men led to a rise in plasma ANP concentrations without a measurable increase in plasma BNP for at least 60 minutes after the load. In contrast, Heringlake et al. [63], using i.v. volume expansion with 15 mL/kg of 0.9% NaCl over 60 minutes, showed that saline infusion induced a brief elevation in plasma NT-pro-ANP followed by a delayed increase in NT-pro-BNP. These and other investigations were all performed in healthy subjects [62, 63]. By comparison, Volpe et al. [64] demonstrated that plasma ANF levels do not rise in response to volume expansion in patients with dilated cardiomyopathy and mild ventricular dysfunction.

Taken together, these studies indicate that an acute saline load in clinically stable individuals is safe and provides a useful tool to probe abnormalities in neurohormonal responses to atrial stretch [62–64]. Experimental data further show that hemodynamic pressure overload provokes a pronounced, though transient, upregulation of proinflammatory cytokines and cytokine receptor genes in the adult heart, supporting the concept that cytokine expression is at least partly load-dependent. Consistent with this notion, our findings suggest that volume depletion induced by high-dose furosemide leads to a marked reduction in circulating cytokine levels that is only partially reversed by subsequent acute volume loading [32].

Collectively, these studies indicate that an acute saline load in clinically stable individuals is safe and represents a useful tool to probe abnormalities in neurohormonal responses to atrial distension [62–64]. Hemodynamic pressure overload has been shown to trigger a marked, albeit transient, upregulation of proinflammatory cytokine and cytokine receptor gene expression in the adult mammalian heart, supporting the concept that cytokine expression is, at least in part, load-dependent. This notion is in line with our findings, which show that volume depletion induced by high-dose furosemide results in a significant reduction in circulating cytokine levels that is only partially reversed by subsequent acute volume loading.

The attenuated percentage change in selected biomarker levels after saline loading in patients treated with high-dose furosemide plus HSS is not straightforward to explain. One plausible hypothesis is that high-dose furosemide combined with HSS, by reducing volume overload and rapidly increasing extracellular NaCl concentration, may relieve myocardial stretch and thereby modulate circulating natriuretic peptides and immuno-inflammatory markers, although the underlying mechanisms remain speculative. To date, no study has specifically examined the effect of HSS on intracellular sodium ([Na⁺]) and its relationship with natriuretic peptide biology. Nonetheless, it is conceivable—pending further experimental and clinical work—that a beneficial effect of HSS on myocardial electrolyte homeostasis may contribute to metabolic protection of the myocardium.

Although the precise mechanism of action of HSS is still not fully defined, several hypotheses have been proposed. Furosemide exerts its diuretic effect after active secretion into the tubular lumen from proximal tubular cells. Many patients with ADHF develop intravascular hypovolaemia and reduced renal blood flow, which impairs this secretion process [48]. Administration of HSS has been shown to increase intraluminal furosemide concentrations and to augment 24-hour diuresis, urinary sodium excretion and urinary osmolality [62, 63]. In addition, HSS has been associated with reductions in plasma renin activity and ANP levels [64].

In the present study, at the compensated stage achieved by both treatment arms, we observed that, after acute saline loading, patients receiving i.v. furosemide + HSS, compared with those treated with furosemide alone, displayed a significantly smaller increase in serum IL-6, hsTnT, sST2, galectin-3 and NT-proBNP. Only a limited number of studies to date have evaluated the impact of i.v. diuretic therapy on neurohormonal and immuno-inflammatory biomarkers. In our setting, the reduction in IL-6 and other markers can reasonably be ascribed to the addition of HSS, which appears to potentiate the effect of furosemide. The transient rise in biomarkers with hemodynamic overload supports the notion that significant cardiomyocyte stretch is occurring. High-dose furosemide plus HSS, by mitigating volume overload and rapidly increasing extracellular NaCl concentrations, may reduce cardiomyocyte stretch and thereby influence NP and immune-inflammatory serum markers [26].

Accordingly, our observation of a less pronounced within-group increase in IL-6, sST2 and NT-proBNP after an acute saline load in patients treated with high-dose furosemide plus HSS, compared with those receiving furosemide alone, is likely related to modulation of the myocardial stretch response to volume overload. We further noted that, at T2 (after saline loading), patients treated for 6 days with i.v. furosemide + HSS showed an increase in IL-6, sST2 and galectin-3 levels relative to T1 (end of i.v. therapy), but these concentrations remained below baseline (T0) values. No significant changes in CRP or hsTnT were detected.

It should also be acknowledged that the higher mean systolic blood pressure observed in the furosemide/HSS group may represent a potential confounder for our results.

The heart is a remarkably plastic organ, capable of undergoing extensive structural and functional remodelling in response to diverse environmental and pathophysiological stimuli [60]. Parallel to these remodelling processes, substantial transcriptional reprogramming takes place in the failing myocardium, and the pivotal contribution of epigenetic regulatory mechanisms in shaping these events has been well recognized [61]. Key components of the epigenetic machinery are biochemically coupled to cellular metabolism through their dependence on specific substrates and cofactors for enzymatic activity [62]. For instance, one study demonstrated that chromatin acetylation machinery can sense alterations in cellular metabolism via acetyl-CoA availability and translate them into transcriptional changes that drive robust phenotypic shifts in pluripotent stem cells [63]. Other biological systems, including stem cells, exhibit similar epigenetic control, whereby a relatively simple metabolic switch can induce widespread genetic reprogramming [64].

Because epigenetic mechanisms are exquisitely sensitive to cellular energetics and mitochondrial function, the epigenetic landscape of the heart under conditions of metabolic and ageing stress undergoes profound changes. From a therapeutic standpoint, these targetable regulators of gene expression hold considerable promise. Indeed, pharmacological modulation of epigenetic modifiers has shown beneficial effects on pathological cardiac remodelling, with reported actions on inflammation, fibrosis, ischaemic injury and hypertrophy [60].

Given that miRNAs can regulate a broad spectrum of target mRNAs, and that individual genes may be modulated by multiple miRNAs, the intricate communication network between these molecules enables miRNAs to influence numerous biological processes, including hypertrophy, fibrosis and angiogenesis, among others. Consequently, miRNAs have substantial potential as therapeutic tools to prevent, attenuate or even reverse cardiac dysfunction, including adverse ventricular remodelling.

In this context, we also documented a more pronounced modulation of HF-related epigenetic signatures, as reflected by changes in selected microRNA fold expression among patients randomized to high-dose furosemide + HSS compared with those receiving i.v. furosemide alone. Specifically, after treatment with high-dose furosemide + HSS, we observed a greater reduction in the fold change of miR-214 and miR-181b* in the combination group than in the furosemide-only group, whereas at T2 a more marked decrease in miR-125a-5p and miR-150-5p* was evident in patients treated with furosemide alone than in those receiving i.v. furosemide + HSS.

MiR-214 has been implicated as a critical regulator of cardiac hypertrophy and maladaptive remodelling pathways involved in HF pathophysiology [65]. Decreased myocardial miR-214 expression has been reported in patients with established cardiac hypertrophy [66, 67]. Moreover, a recent study showed that individuals with systolic HF treated with carvedilol, compared with untreated counterparts, exhibited a significant increase in miR-214 expression [68].

The miR-181 family comprises four members—miR-181a, miR-181b, miR-181c and miR-181d. Although miR-181b has frequently been characterized as an oncogenic miRNA, it has also been associated with vascular inflammatory responses, supporting its role as a modulator of inflammation in HF [69].

MicroRNA-125a-5p (miR-125a-5p) is highly enriched in mesenchymal stromal cell–derived exosomes (MSC-Exos). In a murine I/R model, intramyocardial delivery of miR-125a-5p agomirs, MSCs or MSC-Exos conferred robust cardioprotection, improving cardiac performance and attenuating adverse remodelling. Treatment with miR-125a-5p agomir promoted M2 macrophage polarization, enhanced angiogenesis and reduced fibroblast proliferation and activation, ultimately limiting cardiomyocyte apoptosis and inflammatory responses [68]. Circulating miR-125a-5p has also been proposed as a biomarker of endothelial dysfunction and has been linked to the development of cardiomyopathy [65]. Its expression increases at the onset of reperfusion following ischaemia, where it appears to exert cardioprotective effects by facilitating nitric oxide (NO) signalling [67]. Whether miR-125a-5p mediates MIR4435-2HG–dependent regulatory mechanisms in cardiovascular disease remains to be clarified.

Overall, circulating microRNAs represent highly attractive epigenetic biomarkers because of their biological stability and ease of detection in liquid biopsies. Given the complexity of microRNA-mediated regulation of gene expression and post-transcriptional modification, disease-associated circulating miRNA signatures may mirror underlying pathophysiological states, offering valuable information for distinguishing specific subtypes and stages of complex cardiovascular conditions.

Nevertheless, microRNA serum levels could be considered not only HF diagnostic markers but may also have a possible role as therapeutic indicators.

A prospective, non-randomized self-control trial was performed in 81 patients with HF eligible for CRT to analyze the HF miRNA profile, reporting that HF patients were characterized by lower expression compared to control patients [23]. HF patients considered responders to CRT showed an increase in expression of 19 miRNAs compared with baseline expression, whereas in the non-responders, we observed an increase of six miRNAs compared with baseline expression.

Our findings indicate how the treatment protocol adopted in our study is associated with a higher degree of reduction of change fold of some of the chosen miRNA that have been reported as involved in some of the crucial pathogenetic steps of heart dysfunction such as ventricular hypertrophy and inflammation.

Thus, we have reported that our treatment protocol with high dose i.v. furosemide plus HSS is also characterized by some positive effects also in terms of epigenetic signatures indicating that this type of treatment seems to have on the remodeling mechanisms of the pathogenetic cascade linking to acute systolic left ventricular dysfunction.

miRNAs are involved in the regulation of central elements of the cardiac remodelling cascade, including cardiomyocyte biology and survival, extracellular matrix turnover and neurohormonal signalling. Because miRNAs are coordinately regulated in response to diverse stress stimuli and, in turn, modulate networks of genes that shape the “heart failure phenotype”, it is plausible that individual miRNAs, or specific miRNA clusters, govern the shift from adaptive to maladaptive cardiac remodelling [69–72].

In our cohort, we also detected significantly larger within-group delta (Δ) values at T1–T2—defined as the difference between post-treatment (T1) and post–acute saline load (T2) levels—for miR-21, miR-365 and miR-181b in patients treated with furosemide + HSS or furosemide alone.

MiR-21 is a 22-nucleotide microRNA that binds target mRNAs via complementary base pairing and regulates gene expression by promoting mRNA degradation or inhibiting translation. It has been linked to several forms of cardiomyopathy, including HF, dilated cardiomyopathy, myocardial infarction and diabetic cardiomyopathy.

In these settings, miR-21 expression is markedly altered both in the myocardium and in the circulation, where it appears to confer a degree of cardioprotection following injury, while at the same time being tightly linked to maladaptive processes such as cardiac hypertrophy and fibrosis [70].

A close relationship exists between cardiomyocyte autophagy and the development of cardiac hypertrophy, and persistent stress can drive hypertrophy to progress towards overt HF. MicroRNA-365 (miR-365) has been identified as a positive regulator of cardiac hypertrophy. In a recent study [69], miR-365 expression was found to be upregulated in hypertrophic cardiomyocytes in vivo and in vitro, in parallel with autophagy dysregulation, and miR-365 was shown to negatively modulate autophagic activity in these cells. Collectively, these data support a functional role for miR-365 in the development of cardiac hypertrophy and its transition to systolic dysfunction.

Acute myocardial stretch induced by hemodynamic overload is one of the most common stressors faced by the heart, and the capacity to mount an intrinsic adaptive response is crucial to preventing circulatory congestion. Mechanical stretch stimulates cardiomyocytes to secrete the cardiac natriuretic peptides ANP and BNP, which exert their effects by activating particulate guanylate cyclase A at the cell surface and increasing subsarcolemmal cGMP concentrations [70–75]. The myocardial response to acute stretch thus represents a fundamental adaptive mechanism.

Within this framework, our observation of a blunted rise in inflammatory markers and indices of myocardial stretch after an acute saline load in patients treated with HSS plus moderate/high-dose furosemide suggests that this combination may exert a modulatory effect on these pathways in HFrEF. Such an effect could have important clinical implications, indicating therapeutic benefits that extend beyond mere improvement in congestion-related symptoms and signs. Indeed, the association of i.v. furosemide with HSS may contribute to a “de-remodelling” effect, particularly in terms of attenuating atrial stretch and fibrosis.

This hypothesis could be further sustained and enhanced by our finding related to miRNA change fold after an acute saline load with higher degree of change fold of three miRNA after saline load in controls in comparison to patients treated with high dose furosemide plus HSS further indicating an epigenetic signature of this de-remodeling effect linked to our protocol treatment.

Conclusions

Our results demonstrate that, in patients with ADHF secondary to HFrEF, treatment with moderate-to-high doses of i.v. furosemide in combination with HSS is associated with a significantly greater reduction in circulating IL-6, sST2, hsTnT, galectin-3 and NT-proBNP levels.

Furthermore, we observed that, following an acute saline load, patients receiving high-dose furosemide plus HSS exhibited a smaller rise in several HF-related serum biomarkers, as reflected by lower delta values for the differences in their concentrations before and after volume challenge.