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Background: Aging is associated with metabolic and structural changes causing heart failure with preserved ejection fraction (HFpEF). Interleukin-1 (IL-1) is a pro-inflammatory cytokine involved in aging-related inflammation. We sought to determine whether IL-1 mediates aging-related changes in the heart as seen in HFpEF. Hypothesis: We hypothesize that mice with genetic deletion of IL-1RI, not responsive to IL-1β, would be protected from age-associated cardiac dysfunction. Methods: We studied age-matched young (4-month-old), middle-aged (14-month-old) and old (23-month-old) wild-type (WT) C57BL/6J and IL-1 receptor type I deficient (IL1RI-KO) male mice. Echocardiography was used to left ventricular (LV) dimensions and systolic/diastolic function, and a pressure-transducer was used to measure the LV end-diastolic pressure. Picrosirius red stain was used to assess for myocardial interstitial fibrosis (MIF) at pathology. Results: WT and IL-1RIKO mice showed a normal cardiac phenotype at young age, without any differences in between the two groups. With aging, the WT mice developed LV concentric hypertrophy (as measured by a significant increase in LV mass [+42%, P Conclusions: Genetically-modified mice lacking the IL-1RI receptor, not responsive to IL-1, are protected from aging-related LV hypertrophy, fibrosis and diastolic dysfunction. These data support a central role of IL-1 in the pathophysiology of aging-related HFpEF.

Background: The pleiotropic hormone relaxin has been shown to mediate physiologic responses during pregnancy by facilitating increases in vascular compliance and cardiac output. While relaxin signaling has been attributed to increases in atrial inotropy, the hormonal effects on ventricular contractility have not been explored in vivo . In this study, we investigated the dose-response effects of recombinant human relaxin (serelaxin) on ventricular mechanics. Methods and Results: Adult male CD1 mice were anesthetized and mechanically ventilated prior to Millar cardiac catheterization. A pressure-volume catheter was advanced into the left ventricle (LV), and baseline systolic and diastolic pressure/volume (PV) loops were recorded (Pre). Mice were injected with either saline, 10 μg/kg (low dose) or 1 mg/kg (high dose) of serelaxin, and PV loops were obtained after stabilization (Post) . Mice injected with 1 mg/kg demonstrated significantly higher LV contractility ( Fig A ) independent of preload ( Fig B ). These responses were not observed with low dose. In order to further examine the role of RXFP1 in modulating these responses, healthy CD1 were injected with AAV9-RXFP1 or empty vector as control and receptor overexpression was confirmed four weeks later via qPCR ( Fig C ). PV loop analysis showed an increased inotropic response [ΔP Max , Δ(dP/dt) Max ] and improved relaxation kinetics [Δ(dP/dt) Min , Δτ] in RXFP1-overexpressing mice at a lower dose of serelaxin, when compared to empty vector mice ( Fig D ). Conclusions: Our data show a novel, dose-dependent correlation between serelaxin and ventricular inotropic and lusitropic responses in mice. These responses were observed at a lower drug dose in cardiac RXFP1-overexpressing mice. We propose that further investigation of this phenomenon could advance the therapeutic, cardioprotective effects associated with relaxin therapy in different etiologies of heart failure that may exhibit changes in RXFP1 expression.

Background: Doxorubicin (DOX) is a first-line anticancer drug for the treatment of triple negative breast cancer (TNBC). However, its dose-dependent delayed and progressive cardiotoxicity limits its therapeutic application. NovoMedix (NM922) is a novel dual mTOR inhibitor/AMPK activator that was shown to attenuate adverse cardiac remodeling and fibrosis in a pressure-overload mouse model of heart failure. We investigated whether combination therapy with DOX and NM922 exhibits synergistic chemotherapeutic effect while mitigating DOX cardiotoxicity. Methods & Results: Tumors were generated in athymic female BALB/cAnNCr-nu/nu mice by implanting MDA-MB-231 cells into the rear right flank. Mice with tumors (volume≈200mm 3 ) were randomized into 6 groups and treated as follows: 1) Control (n=10); 2) DOX (3 mg/kg; i.p. twice weekly, total 15 mg/kg; n=10); 3) NM922 (25 mg/kg/d; p.o. n=5); 4) DOX+NM922 (25 mg/kg/d; p.o. n=15); 5) NM922 (100 mg/kg/d; p.o. n=5); 6) DOX+NM922 (100 mg/kg/d; p.o. n=15). Tumor size, body weight and cardiac function were assessed throughout the study. DOX alone, and to a significant extent when in combination with NM922 (25 mg/kg) reduced tumor growth compared to control. NM922 (100 mg/kg) with/without DOX significantly reduced tumor growth as compared to DOX alone (Fig A). DOX caused reduction in body weight and survival of tumor-bearing mice. NM922 did not prevent DOX-induced cachexia, but significantly improved survival in DOX-treated mice (Fig B). DOX treatment caused a significant decline in left ventricular ejection fraction compared to control over 3 weeks, which was ameliorated with NM922 (100 mg/kg) co-treatment (Fig C&D). Conclusion: Our results suggest that NM922 may potentiate the chemotherapeutic efficacy of DOX in TNBC, while mitigating its cardiotoxicity. Moreover, these findings advocate the potential efficacy of utilizing lower DOX dosages when combined with NM922, which would have significant clinical implications.

Background: Sacubitril/Valsartan (SAC/VAL), a drug combining a neprilysin inhibitor and an angiotensin receptor blocker, was shown to reduce myocardial infarct size and left ventricular (LV) dysfunction in preclinical models of myocardial infarction (MI). In the PARADIGM-HF trial, SAC/VAL prevented the clinical progression of patients with heart failure (HF) more effectively than enalapril. Whether SAC/VAL attenuates cardiac fibrosis and improves LV function in a rabbit model of MI-induced HF with reduced ejection fraction (rEF) is unknown. Methods: Anesthetized adult male NZW rabbits (~2.5kg) underwent left thoracotomy and the left anterior descending (LAD) coronary artery was identified and occluded for 45 min followed by reperfusion. Weekly echocardiography was performed to confirm reduced EF (~40%), which was uniformly achieved at 5 weeks post MI. Subsequently, rabbits were randomized to orally receive placebo (volume-matched water, BID), SAC/VAL (10 mg/kg, BID) or VAL (9.1mg/kg/day) starting on week 6. At 10 weeks post MI, rabbits were sacrificed and hearts were harvested, fixed with 10% formalin and embedded in paraffin to assess myocardial fibrosis (Picrosirius red staining). Operators performing echocardiography, Picrosirius red staining and analysis were blinded to treatment allocation. Results: Two weeks after treatment initiation, a significant improvement in LVEF was observed in the SAC/VAL group compared to both placebo and VAL, a benefit that lasted throughout the entire study (Fig. A). The functional improvement observed was associated with a significant reduction in LV scar size compared to placebo at week 10 (Fig. B). However, when compared to VAL, the decrease in scar size did not reach statistical significance despite a clear trend. Conclusion: Our results suggest that SAC/VAL may offer superior benefits compared to equivalent dose of stand-alone VAL in attenuating LV scar size and improving LVEF in a rabbit model of ischemic HFrEF.

Background: Hydrogen sulfide (H2S) has been shown to attenuate myocardial ischemia/reperfusion injury via suppression of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Whether the H2S donor, Na2S, protects against ischemic heart failure with reduced ejection fraction (HFrEF) when treatment is initiated after development of LV dysfunction is unknown. Methods and Results: Adult male mice underwent myocardial infarction (MI) by permanent coronary artery ligation after baseline echocardiography. Repeat echocardiography was performed at day 3 post MI and surviving mice with fractional shortening (FS) less than 25% were treated with either Na2S (100 μg/kg, ip) or saline (volume matched, ip) for 25 days. LV fractional shortening remained unchanged at 7 and 28 days post-MI in the saline group, but improved significantly with Na2S at both time points (Fig. A). Moreover, LV infarct scar size, assessed by trichrome staining, was smaller in Na2S group (14.8 ± 2.1%) as compared to control (28.8 ± 4.8%, P Conclusion: Treatment with Na2S in mice with ischemic HFrEF improves LV function and survival up to 28 days post MI, possibly through suppression of ASC and prevention of further NLRP3 inflammasome formation. We propose that H2S donors can be promising therapeutic tools for ischemic HF.

Introduction: Radiation therapy (XRT) used for the treatment of cancer, can expose the heart to the radiation beam causing cardiac injury. However, fatigue and exercise intolerance are common symptoms in patients treated with XRT for different types of cancer, in which the XRT field does not involve the heart. Hypothesis: We hypothesize that impaired cardiac reserve may occur even when the XRT field is distant from the heart (‘out of target effect’). Methods: C57BL/6J female mice underwent two different radiation treatments (20 Gray, single dose) using a Small Animal Radiation Research Platform: a) the isocenter was pointed to the right lung sparing the heart (‘out of target’) and b) the heart was chosen as isocenter, as positive control. An additional group of mice were sham-irradiated, as negative control. Transthoracic echocardiography was used to assess the left ventricular ejection fraction (LVEF) at baseline and the contractile reserve expressed as percentage change in the LVEF measured at rest (LVEFr) and 3 minutes after intraperitoneal administration of the β-adrenergic agonist, isoproterenol (LVEFi)[(LVEFi-LVEFr)/LVEFr]*100]. Results: Three days after radiation treatment irradiated and sham mice showed normal LVEF at rest. However, the ‘out of target’ irradiation group showed a significant impairment in cardiac contractile reserve compared with sham, and similar to the ‘on target’ group (Figure). Conclusions: ‘Out of target’ radiation therapy leads to impaired cardiac contractile reserve in the mouse.

Background: The preconditioning-like infarct-sparing and anti-inflammatory effects of the peptide hormone relaxin following ischemic injury have been studied in the heart. Whether reperfusion therapy with recombinant human relaxin (serelaxin, SRLX) reduces myocardial infarct size and attenuates NLRP3 inflammasome formation/caspase-1 activation and subsequent loss of functional myocardium following ischemia/reperfusion (I/R) injury is unknown. Methods and Results: After baseline echocardiography, adult male C57BL (WT) or eNOS knockout (KO) mice underwent myocardial infarction (MI) by coronary artery ligation for 30 minutes followed by 24 h reperfusion. Mice were treated with either SRLX (10 μg/Kg; sc) or saline 5 minutes before reperfusion. SRLX improved survival at 24 h post MI in WT mice (79%) as compared with controls (42%), whereas there was no difference in survival between SRLX- and saline-treated eNOS KO mice. Moreover, SRLX significantly reduced infarct size, measured with TTC staining, and preserved LV fractional shortening (FS) and end-systolic diameter (LVESD) in WT mice as compared with controls. Interestingly, cardiac caspase-1 activity was markedly reduced in SRLX-treated mice compared with controls at 24 h post MI (Figure A-D). Genetic deletion of eNOS abolished the infarct-sparing and anti-inflammatory effects of SRLX as well as functional preservation. SRLX plasma levels were assessed 5 min. after treatment using ELISA and the results demonstrate therapeutic levels comparable to plasma relaxin during the first trimester of pregnancy (Figure E). Conclusion: Reperfusion therapy with SRLX attenuates myocardial I/R injury and NLRP3 inflammasome formation via eNOS-dependent mechanism. We propose that SRLX possesses an anti-inflammatory effect preventing caspase-1 activation and inflammatory complications following MI, which may shed some light on the mechanism behind the survival benefit observed in the RELAX-AHF trial.

Introduction: The NLRP3 inflammasome is activated in the heart following ischemic injury, and it promotes cardiac dysfunction. Ischemic injury establishes both a priming signal (leading to transcription of inflammasome components) and a trigger (NLRP3 activation). Whether activation of NLRP3 (in absence of priming) represents the limiting step in the formation of the inflammasome in the heart is unknown. Hypothesis: Both priming and the triggering signals in the heart are necessary for the formation of the inflammasome and ensuing cardiac dysfunction. Methods: We induced systemic expression of a floxed mutant NLRP3-A350V, that is constitutively active, in 10 mice using a tamoxifen-inducible Cre recombinase, to create a condition in which NLRP3 is activated, in absence or presence of priming with low dose LPS (2 mg/kg). Molecular analyses were performed on the heart and the spleen (myeloid organ not dependent on priming) to measure caspase-1 activity using a fluorogenic assay, mRNA expression of the component of the inflammasome (caspase-1 and IL-1β), and echocardiography to measure left ventricular (LV) dimension and systolic function. Results: NLRP3-A350V mutant mice had increased caspase-1 activity in the spleen but not in the heart and had normal LV systolic function (Figure), showing that in NLRP3 activation without priming is insufficient to induce inflammasome formation in the heart nor LV systolic dysfunction. Priming with LPS induced the expression of the inflammasome components in the heart which in absence of NLRP3 activation (control mouse) had no effects on LV systolic function, whereas in the presence of NLRP3 activation (mutant mouse) led to reduced LV systolic function and premature death (Figure). Conclusions: The inflammasome formation in the heart requires a priming signal to be coupled with activation of NLRP3 in order to induce LV dysfunction.