Decrease In Fractional Shortening Research Articles

Ambulatory electrocardiographic longitudinal monitoring in a canine model for Duchenne muscular dystrophy identifies decreased very low frequency power as a hallmark of impaired heart rate variability.

Duchenne muscular dystrophy (DMD) patients exhibit a late left ventricular systolic dysfunction preceded by an occult phase, during which myocardial fibrosis progresses and some early functional impairments can be detected. These latter include electrocardiographic (ECG) and heart rate variability (HRV) abnormalities. This longitudinal study aimed at describing the sequence of ECG and HRV abnormalities, using Holter ECG in the GRMD (Golden retriever muscular dystrophy) dog model, known to develop a DMD-like disease, including cardiomyopathy. Most of the known ECG abnormalities described in DMD patients were also found in GRMD dogs, including increased heart rate, prolonged QT and shortened PR intervals, ventricular arrhythmias, and several of them could be detected months before the decrease of fractional shortening. The HRV was impaired like in DMD patients, one of the earliest evidenced abnormalities being a decrease in the very low frequency (VLF) component of the power spectrum. This decrease was correlated with the further reduction of fractional shortening. Such decreased VLF probably reflects impaired autonomic function and abnormal vasomotor tone. This study provides new insights into the knowledge of the GRMD dog model and DMD cardiomyopathy and emphasizes the interest to monitor the VLF power in DMD patients, still unexplored in this disease, whilst it is highly predictive of deleterious clinical events in many other pathological conditions.

Sufentanil reduces myocardial apoptosis in rats with myocardial ischemia-reperfusion injury

Purpose: To determine the effect of sufentanil on myocardial apoptosis in rats with myocardial ischemia-reperfusion injury (MIRI).Methods: Fifty Sprague Dawley rats were randomly assigned to five groups: sham, model, low-dose, moderate-dose, and high-dose. The groups, except sham, underwent ligation of the left anterior descending coronary artery to establish the MIRI model. The low, moderate, and high-dose groups received intraperitoneal injections of sufentanil at different concentrations. Cardiac function, serum LDH, creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA) content were evaluated. The mRNA expression levels of apoptosis genes and protein levels of p38 and p-p38 were assessed in myocardial tissues using various methods while apoptosis was assessed by TUNEL assay.Results: Compared to sham group, the model group exhibited significant decrease in fractional shortening (FS) and ejection fraction (EF), increase in CK activity, LDH, and MDA contents, lower SOD activity. Model group also showed increase in mRNA levels of B-cell lymphoma-2 (Bcl-2) and caspase- 3, higher apoptosis, significant increase in protein levels of p38 and p-p38, and higher level of myocardial apoptosis (p < 0.05). High-dose group demonstrated significant increase in FS and EF, decrease in LDH content and CK activity, lower MDA content, higher SOD activity, decrease in mRNA levels of Bcl-2 and caspase-3, lower apoptosis, decrease in protein levels of p38 and p-p38, and lower level of myocardial apoptosis (p < 0.05), when compared with model group.Conclusion: High-dose sufentanil reduces myocardial apoptosis and improves cardiac function, and thus can potentially be developed as a cardioprotective agent.

PS-BPB02-4: LEFT VENTRICLE HYPERTROPHY AND DYSFUNCTION WERE EXAGGERATED BY P2RX7 KNOCKOUT BUT UNAFFECTED BY P2RX7 PHARMACOLOGIC BLOCKADE IN ANGIOTENSIN II-INFUSED MICE

Objective: Extracellular adenosine triphosphate released from damaged cells binds to the P2X7 receptor (P2RX7) to activate of immune cells. Our preliminary data demonstrated that P2RX7 plays a role in vascular injury of hypertensive mice. Angiotensin (Ang) II-induced hypertension, vascular injury and aortic perivascular infiltration of activated T cell were attenuated by P2rx7 knockout or P2RX7 pharmacologic blockade. However, whether P2RX7 is involved in Ang II-induced cardiac dysfunction and hypertrophy is unknown. We hypothesized that Ang II-induced left ventricular (LV) dysfunction and hypertrophy would be blunted by P2rx7 knockout or P2RX7 antagonism. Design and method: Ten-to-12-week-old male C57BL/6J wild-type (WT) and P2rx7-/- mice were infused or not with Ang II (1000 ng/kg/min) for 14 days. A second set of Ang II-infused WT mice was infused with the P2RX7 antagonist AZ10606120 (694 ng/kg/min) or vehicle for 14 days. Cardiac LV function and remodeling were determined by ultrasound. Total RNA was isolated from the heart using the mirVana miRNA isolation kit. Relative expression was calculated using the Pfaffl method with 40S ribosomal protein S16 (Rps16) as a reference gene. Hypertrophic and fibrotic markers were studied by reverse transcription-quantitative PCR (RT-qPCR) in cardiac ventricles. Results: Ang II-induced 1.5-fold increase in LV mass/body weight (P < 0.001) and ∽25% decrease in fractional shortening in WT mice and mice receiving AZ10606120 (P < 0.05). These changes were exaggerated in P2rx7-/- mice (LV mass/body weight: 1.7-fold increase, fractional shortening: 54% decrease, P < 0.05), but not in mice receiving AZ10606120. Ang II-induced atrial natriuretic peptide (Nppa/Rps16: 7-fold, P < 0.001) and α-skeletal actin 1 expression (Acta1/Rps16: 6-fold, P < 0.001) tended to be further increased in P2rx7-/- mice (Nppa/Rps16: 14-fold and Acta1/Rps16: 12-fold), but not in AZ10606120-treated mice. Ang II-induced myosin heavy chain 7B (14-fold, P < 0.05) was unaffected by P2rx7 knockout or P2RX7 antagonism. Ang II caused a 1.5-fold increase in transforming growth factor β; 1 only in P2rx7-/- mice (P < 0.05). Conclusion: Ang II-induced LV hypertrophy and dysfunction were exaggerated by P2rx7 knockout but were not affected by P2RX7 pharmacologic blockade.

Cardioprotective effects of preconditioning exercise in the female tumor bearing mouse.

Cancer cachexia, a metabolic wasting syndrome, affects up to 80% of cancer patients and leads to the death in up to 20% of cancer patients. While research is growing in the field, there are still no clear diagnostic criteria and cancer cachexia remains an untreated condition. Aerobic exercise has been shown to positively impact cachexia by slowing its development and attenuating muscle loss. The most effective timing, duration, and intensity of exercise as a preventative and protective measure against cancer cachexia remains questionable. Therefore, the purpose of this study was to examine the effects of preconditioning exercise as a protective measure for tumor-mediated muscle wasting. Female LC3 Tg+ and wildtype mice were randomly separated into four groups, sedentary non-tumor bearing (SED + NT), sedentary tumor bearing (SED + T), treadmill exercise non-tumor bearing (TM + NT), and treadmill exercise tumor bearing (TM + T). Mice underwent an 8-week treadmill exercise training protocol (TM) or remained sedentary (SED). Next, mice were implanted with tumor cells (T group; 5 × 105 Lewis Lung Carcinoma cells in flank) or remained non-tumor (NT) for 4 weeks. Tumor bearing resulted in a significant decline in cardiac function. SED + T showed a significant decrease in fractional shortening (p < 0.05) when compared to the other groups. This coincided with an increase in beclin-1 and MyD88 protein expression and decrease in p-FOXO1 (inactivated) protein expression in SED + T mice. Interestingly, preconditioning exercise (exercise prior to tumor bearing) appeared to preserve cardiac function (TM + T not significantly different than SED + NT). Exercise-mediated cardioprotection also coincided with abolished beclin-1 and MyD88 signaling that was not significantly elevated in TM + T mice. Additionally, TM resulted in a 22-fold decrease in estimated tumor volume (p < 0.05) and a 45% decrease in tumor mass (p < 0.05) compared to SED tumors. The data indicate potential cardioprotective effects of preconditioning exercise on preserving cardiac structure and function, as well as regulating autophagic (beclin-1), inflammatory (TGF-β and MyD88), and atrophy (p-FOXO1) pathways during tumor bearing. Preconditioning exercise may be an effective and accessible treatment intervention for early-stage cancer survivors. This data is crucial in identifying the significance of exercise and the timing of exercise as a protective measure against the detrimental effects of cancer cachexia.

Abstract P316: ANGIOTENSIN II-INDUCED LEFT VENTRICLE HYPERTROPHY AND DYSFUNCTION WERE EXAGGERATED BY P2RX7 KNOCKOUT BUT UNAFFECTED BY P2RX7 PHARMACOLOGIC BLOCKADE

Introduction: We have demonstrated that the P2X7 receptor (P2RX7), which activates immune cells via binding of extracellular adenosine triphosphate, plays a role in hypertension and vascular injury. Angiotensin (Ang) II-induced hypertension, vascular injury and perivascular infiltration of activated CD8 + T cells were attenuated by P2rx7 knockout or P2RX7 pharmacologic blockade. However, whether P2RX7 is involved in Ang II-induced cardiac dysfunction and hypertrophy remains unknown. Hypothesis: We hypothesized that Ang II-induced left ventricular (LV) dysfunction and hypertrophy will be blunted equally by P2rx7 knockout and P2RX7 antagonism. Methods: Ten-to-12-week-old male C57BL/6J wild-type (WT) and P2rx7 -/- mice were infused or not with Ang II (1000 ng/kg/min) for 14 days. A second group of WT mice infused with Ang II was infused with the P2RX7 antagonist AZ10606120 (694 ng/kg/min) or vehicle for 14 days. Cardiac LV function and remodeling were determined by ultrasound, and hypertrophic markers in cardiac ventricles by reverse transcription-quantitative PCR (RT-qPCR). Results: Ang II-induced increase in LV mass/body weight (6.3±0.2 vs 4.2±0.2 mg/g, P &lt;0.001) and decrease in fractional shortening in WT mice (32.5±3.1% vs 43.8±2.4%, P &lt;0.05) were exaggerated in P2rx7 -/- mice (7.3±0.5 mg/g and 20.2±3.1%, P &lt;0.05), but not in mice receiving AZ10606120 (6.2±0.4 mg/g and 28.9±3.3%). Ang II-induced atrial natriuretic peptide ( Nppa /ribosomal protein S16 [ Rps16 ], 6.8±2.3 vs 1±0.1, P &lt;0.001) and α-skeletal actin 1 expression ( Acta1 / Rps16 , 6.3±1.3 vs 1±0.2, P &lt;0.001) tended to be further increased in P2rx7 -/- mice ( Nppa / Rps16 : 13.5±2.4 and Acta1 / Rps16 : 11.9±2.4), but not in AZ10606120-treated mice ( Nppa / Rps16 : 5.5±0.7 and Acta1 / Rps16 : 5.6±0.8). Conclusion: Ang II-induced LV hypertrophy and dysfunction were exaggerated by P2rx7 knockout but were not affected by P2RX7 pharmacologic blockade.

Preconditioning Exercise Protects Against Tumor‐Mediated Muscle Dysfunction in the Male Mouse

BackgroundCancer is associated with cardiac and skeletal muscle wasting. While exercise has been shown to be myoprotective, less is known about the most effective “dose” of exercise in terms of intensity, timing, and duration. Therefore, the purpose of this study was to determine the effects of preconditioning exercise – exercise completedJ before tumor bearing ‐ as a protective measure for tumor‐mediated muscle wasting.MethodsMale mice were randomly separated into four groups: sedentary non‐tumor bearing (SED+NT), sedentary tumor bearing (SED+T), treadmill exercise non‐tumor bearing (TM+NT), and treadmill exercise tumor bearing (TM+T). Mice underwent an 8‐week progressive treadmill exercise training protocol (TM) or remained sedentary (SED). At the end of the preconditioning treadmill training protocol, mice remained sedentary for 4 weeks. During this 4 week sedentary period, mice were implanted with tumor cells (T group; 1x105 LLC cells in flank) or remained non‐tumor (NT). To determine whether 8 weeks of exercise preconditioning could preserve cardiac and skeletal muscle function, mice underwent echocardiogram and grip strength analyses at baseline, 8‐week, and 12‐week time points.ResultsTumor bearing resulted in significant muscle dysfunction. SED+T showed a significant decrease in fractional shortening (p&lt;0.05) and grip strength (p&lt;0.05) when compared to all other groups. This coincided with SED+T’s significantly smaller heart masses and skeletal masses (p&lt;0.05 vs to all other groups). Interestingly, preconditioning exercise (exercise prior to tumor bearing) appeared to offer myoprotection since TM groups were not significantly different from SED+NT for fractional shortening and grip strength (p&gt;0.05). This data was in step with preserved heart mass and skeletal muscle mass in TM+T (p&gt;0.05 vs SED+NT).ConclusionThese data indicate that exercise initiated prior to tumor bearing can protect the musculature from tumor‐mediated muscle dysfunction. This may be attributed to slowed muscle wasting as evidenced by maintenance of muscle mass in TM+T groups. These data make an important case for the long‐term benefits of an active lifestyle that last even after exercise is stopped (i.e., upon diagnosis and treatment). Such information is critical in elucidating the most effective “dose” of exercise for cancer survivors.

Cardioprotective Effects of Preconditioning Exercise in the Female Tumor‐Bearing Mouse

BackgroundCancer cachexia, a metabolic wasting syndrome, affects up to 80% of cancer patients and leads to the death in up to 20% of cancer patients. While research is growing in the field, there are still no clear diagnostic criteria and cancer cachexia remains an untreated condition. Aerobic exercise has been shown to positively impact cachexia by slowing its development and attenuating muscle loss. The most effective timing, duration, and intensity of exercise as a preventative and protective measure against cancer cachexia remains questionable. Therefore, the purpose of this study was to examine the effects of preconditioning exercise as a protective measure for tumor‐mediated muscle wasting.MethodsFemale LC3 Tg+ and WT mice were randomly separated into four groups, sedentary non‐tumor bearing (SED+NT), sedentary tumor bearing (SED+T), treadmill exercise non‐tumor bearing (TM+NT), and treadmill exercise tumor bearing (TM+T). Mice underwent an 8‐week treadmill exercise training protocol (TM) or remained sedentary (SED). Next, mice were implanted with tumor cells (T group; 1x105 LLC cells in flank) or remained non‐tumor (NT) for 4 weeks. To examine the protective effects of exercise on tumor growth and muscle wasting during tumor‐mediated muscle wasting, grip strength and tumor growth evaluations were taken at baseline, 8‐week, and 12‐week time points. At the end of the 12‐week study, muscle and tumor tissue was collected and weighed.ResultsTumor bearing resulted in a significant decline in cardiac function. SED+T showed a significant decrease in fractional shortening (P &lt; 0.05) when compared to the other groups. This coincided with an increase in beclin‐1 expression in SED+T mice. Interestingly, preconditioning exercise (exercise prior to tumor bearing) appeared to preserve cardiac function (TM+T not significantly different than SED+NT). Exercise‐mediated cardioprotection also coincided with abolished beclin‐1 signaling since this was not significantly elevated in TM+T mice. Additionally, TM resulted in a 20‐fold decrease in estimated tumor volume (P &lt; 0.05) and a 60% decrease in tumor mass (P &lt; 0.05) compared to SED tumors.ConclusionThe data indicate potential cardioprotective effects of preconditioning exercise on preserving cardiac structure and function, as well as regulating autophagic pathways (beclin‐1) during tumor bearing. Preconditioning exercise may be an effective and accessible treatment intervention for early‐stage cancer survivors. This data is crucial in identifying the significance of exercise and the timing of exercise as a protective measure against the detrimental effects of cancer cachexia.

Pharmacogenomic study of anthracycline-induced cardiotoxicity in Mexican pediatric patients.

Background: The aim of this study was to evaluate the association between well-defined genetic risk variants in SLC28A3, RARG and UGT1A6 and anthracycline-induced cardiotoxicity in Mexican pediatric patients. Methods: We tested a cohort of 79 children treated with anthracyclines for the presence of SLC28A3-rs7853758, RARG-rs2229774 and UGT1A6-rs17863783. Results: The SLC28A3-rs7853758 variant was more frequent in this cohort, while the UGT1A6-rs17863783 and RARG-rs2229774 variants were present at lower frequencies. A clinically important decrease of fractional shortening was associated with SLC28A3-rs7853758 variant. Conclusion: In this cohort, 39.2% of patients carried the protective SLC28A3 variant. A small number of tested patients have the risk variants of UGT1A6 and RARG. None of the patients shared the two risk variants.

Abstract 10912: Sarpogrelate, an Antiplatelet Agent, Suppressed Cardiac Hypertrophy and Systolic Dysfunction in a 5-ht 2a Receptor-Independent Manner

Introduction: Recently, the cost of new drug development is increasing year by year, and drug repositioning is being used as a strategy for developing new treatments by saving time and costs. We used a library of approved drugs to screen for compounds that suppress cardiomyocyte hypertrophy and identified the antiplatelet drug sarpogrelate, a selective serotonin-2A receptor antagonist, as a candidate for heart failure therapy. In this study, we investigated the effect of sarpogrelate on cultured cardiomyocyte hypertrophy and development of heart failure. Methods &amp; Results: First, primary cultured cardiomyocytes were treated with 1 μM sarpogrelate and then stimulated with various hypertrophic stimuli (30 μM phenylephrine (PE), 0.1 μM angiotensin II and 0.1 μM endothelin 1). Sarpogrelate repressed hypertrophic responses induced by each stimulus, such as myofibrillar organization and increase in cell size. Moreover, PE-induced transactivation of the hypertrophic response genes promoter was significantly inhibited by sarpogrelate. Western blotting (WB) showed that sarpogrelate significantly suppressed PE-induced the phosphorylation of ERK1/2 and GATA4. Next, C57BL/6j male mice were subjected to a transverse aortic constriction (TAC) and sham operation. One day after the operation, the mice were randomly divided into 3 groups: sarpogrelate at 1 mg/kg or 5 mg/kg, and vehicle as a control. Daily oral administration was repeated for 8 weeks. Echocardiographic analysis showed that 5 mg/kg sarpogrelate significantly prevented a TAC-induced increase in posterior left ventricular wall thickness and a decrease in fractional shortening at 8 weeks after the operation. Five mg/kg sarpogrelate also suppressed a TAC-induced increase in HW/BW ratio, myocardial cell diameter, perivascular fibrosis, and mRNA levels of ANF and BNP. Moreover, the WB analysis showed that 5 mg/kg sarpogrelate significantly suppressed TAC-induced phosphorylation of the ERK1/2 and GATA4. Conclusions: Sarpogrelate significantly suppresses cardiomyocyte hypertrophy and the development of heart failure via at least, in part, by inhibition of ERK1/2-GATA4 pathway. These findings suggest that sarpogrelate may be an effective agent for heart failure therapy.

Chronic Benzene Exposure Aggravates Pressure Overload-Induced Cardiac Dysfunction.

Benzene is a ubiquitous environmental pollutant abundant in household products, petrochemicals, and cigarette smoke. Benzene is a well-known carcinogen in humans and experimental animals; however, little is known about the cardiovascular toxicity of benzene. Recent population-based studies indicate that benzene exposure is associated with an increased risk for heart failure. Nonetheless, it is unclear whether benzene exposure is sufficient to induce and/or exacerbate heart failure. We examined the effects of benzene (50 ppm, 6 h/day, 5 days/week, and 6 weeks) or high-efficiency particulate absorbing-filtered air exposure on transverse aortic constriction (TAC)-induced pressure overload in male C57BL/6J mice. Our data show that benzene exposure had no effect on cardiac function in the Sham group; however, it significantly compromised cardiac function as depicted by a significant decrease in fractional shortening and ejection fraction, as compared with TAC/Air-exposed mice. RNA-seq analysis of the cardiac tissue from the TAC/benzene-exposed mice showed a significant increase in several genes associated with adhesion molecules, cell-cell adhesion, inflammation, and stress response. In particular, neutrophils were implicated in our unbiased analyses. Indeed, immunofluorescence studies showed that TAC/benzene exposure promotes infiltration of CD11b+/S100A8+/myeloperoxidase+-positive neutrophils in the hearts by 3-fold. In vitro, the benzene metabolites, hydroquinone, and catechol, induced the expression of P-selectin in cardiac microvascular endothelial cells by 5-fold and increased the adhesion of neutrophils to these endothelial cells by 1.5- to 2.0-fold. Benzene metabolite-induced adhesion of neutrophils to the endothelial cells was attenuated by anti-P-selectin antibody. Together, these data suggest that benzene exacerbates heart failure by promoting endothelial activation and neutrophil recruitment.

Ipilimumab and Nivolumab exertes cardiotoxic and pro-fibrotic effects in mice through the overexpression of NLRP3 inflammasome, chemokines and leukotrienes

Abstract Background Several strategies based on immune checkpoint inhibitors (ICIs) have been developed for cancer therapy, opening to advantages in cancer outcomes. However, several ICIs-induced side effects emerged in these patients, especially a rare but clinically significant cardiotoxicity with high rate of mortality. Purpose We studied cytotoxic and pro-inflammatory properties of Ipilimumab and Nivolumab in cellular and preclinical models Methods Co-cultures of human cardiomyocytes and hPBMCs were exposed to Ipilimumab or Nivolumab at 100 nM; cell viability and expression of leukotrienes, NLRP3 inflammasome, MyD88 (myddosome) and p65/NF-kB were performed. C57 mice were untreated (Sham; n=6) or treated with Ipilimumab (IPI, n=6) (15 mg/kg); analysis of fractional shortening, ejection fraction, radial and longitudinal strain were made before and after treatments through 2D-echocardiography (Vevo 2100). Expression of NLRP3, MyD88, p65/NF-kB, leukotrienes and 12 cytokines (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL17-α, IFN-γ, TNF-α, G-CSF, and GM-CSF) have been analyzed in murine myocardium. Results Nivolumab and Ipilimumab induced cell death and apoptosis in cardiomyocutes. Both ICIs increased NLRP3, MyD88 and p65/NF-kB expression compared to untreated cells, however the most pro-inflammatory and cardiotoxic effects were seen after exposure to Ipilimumab. Mice treated with Ipilimumab showed a significant decrease of fractional shortening and radial strain compared to untreated mice. Metabolic studies clearly indicates that ipilimumab increases leukotrienes production and NLRP3 expression in heart tissues, probably due to increased iROS content (iROS are key inductors of leukotrienes expression). High expression of IL-6, IL-1 and IL-17 were also seein in mice treated with ipilimumab (p&amp;lt;0,001). Conclusions Nivolumab and Ipilimumab exert cytotoxic effects mediated by NLRP3 inflammasome, leukotrienes and pro-inflammatory cytokines in heart tissues. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministero della Salute, Ricerca Corrente Project

Abstract P348: Pathophysiological Basis Of A Compound Variant In Calcium And Sarcomere Regulation Causing Cardiac Arrhythmias And Hypertrophic Cardiomyopathy

Rationale: Hypertrophic cardiomyopathy (HCM) is common inheritable heart disease. HCM is highly associated with arrhythmias and/or sudden death. Studies show that molecular defects in calcium handling impairing the cardiomyocyte contractility is a predominant cause. However, the pathophysiology underlying HCM with arrhythmias is not well understood, hindering the identification of novel therapies. Objective: To investigate the pathophysiological consequences of compound variants, consisting of Histidine Rich Calcium Binding Protein gene ( HRC S96A ) and an intronic 25bp deletion in cardiac myosin binding protein-C ( MYBPC3 Δ25bp ). Methods and Results: Clinical data revealed that co-segregation of HRC S96A and MYBPC3 ΔInt32 results in cardiac arrhythmia, heart failure, and sudden cardiac death in South Asians. To determine the cellular/molecular trigger underlying the pathophysiology of this dual variant, we used humanized, knock-in, heterozygous mouse models, including HRC S81A (equivalent to HRC S96A ) MYBPC3 Δ25bp , HRC S81A / MYBPC3 Δ25bp (double variant, DV), and wild-type controls. Echocardiography revealed a significant decrease in the percentage of ejection fraction and fractional shortening in DV mice, as well as the presence of diastolic dysfunction, at 12 weeks of age, compared to single-variant and wild-type mice. Electrocardiogram tracing of DV mice showed the presence of stress-induced arrhythmias, such as ventricular tachycardia after caffeine and epinephrine administration. Using isolated cardiomyocytes in vitro , Calcium transient experiments indicated a significant decrease in fractional shortening, Ca 2+ transient amplitude, and a higher number of after-contractions in cardiomyocytes from DV mice. DV mouse hearts showed increased phosphorylation of CaMKII and SR Ca 2+ leak by cardiomyocytes. Inclusion of the CaMKII inhibitor KN-93 rescued the increases in SR Ca 2+ leak and in aftercontractions. Conclusion: Impaired Ca 2+ -handling, owing to the HRC S96A variant, aggravates SR Ca 2+ leak and aftercontractions in MYBPC3 Δ25bp cardiomyocytes, subsequently triggering cardiac arrhythmias and sudden death in vivo .

Abstract P352: An Antiplatelet Agent Sarpogrelate Suppresses Pressure Overload-induced Development Of Heart Failure In A 5-ht 2a Receptor-independent Manner

Purpose: The cost of new drug development is increasing year by year, and drug repositioning is being used as a strategy to develop new treatments at low-cost. We used a library of approved drugs to screen for compounds that suppress cardiomyocyte hypertrophy, and identified as a candidate the antiplatelet drug sarpogrelate, a selective serotonin-2A (5-HT 2A ) receptor antagonist. In this study, we examined the effect of sarpogrelate on cultured cardiomyocyte hypertrophy and development of heart failure. Methods &amp; Results: First, primary cultured cardiomyocytes were treated with 1 μM sarpogrelate and then stimulated with various hypertrophic stimuli (30 μM phenylephrine (PE), 0.1 μM angiotensin II and 0.1 μM endothelin 1). The results of immunofluorescence staining with anti-MHC antibody showed that sarpogrelate significantly suppressed cardiomyocyte hypertrophy induced by each stimulus. Western blotting and qPCR analysis showed that the mRNA and protein levels of 5-HT 2A receptor did not change by PE, and sarpogrelate significantly suppressed PE-induced phosphorylation of ERK1/2 and GATA4. Next, C57BL/6j male mice were subjected to a transverse aortic constriction (TAC) and sham operation. One day after the operation, the mice were randomly divided into 3 groups: sarpogrelate at 1 mg/kg or 5 mg/kg, and vehicle as a control. Daily oral administration was repeated for 8 weeks. Echocardiographic analysis showed that 5 mg/kg sarpogrelate significantly prevented a TAC-induced increase in posterior left ventricular wall thickness and a decrease in fractional shortening at 8 weeks after the operation. Five mg/kg sarpogrelate also suppressed TAC-induced increase in HW/BW ratio, cross-sectional areas, perivascular fibrosis, and mRNA levels of ANF and BNP. Moreover, the western blotting analysis showed that 5 mg/kg sarpogrelate significantly suppressed TAC-induced phosphorylation of ERK1/2. Conclusions: These results indicate that sarpogrelate significantly suppresses cardiomyocyte hypertrophy and the development of heart failure via at least, in part, by inhibition of ERK1/2-GATA4 pathway. These findings suggest that sarpogrelate may be an effective agent for heart failure therapy.

Abstract MP217: Nuclease-deficient Cas9 Transcription Factors Restore Krueppel-like Factor 15 Expression In Stressed Mouse And Human Cardiomyocytes

Transcriptional changes in cardiomyocytes drive heart failure progression, however, precise control over endogenous gene expression remains challenging. The expression of Krueppel-like factor 15 ( KLF15 ), an evolutionary conserved nuclear and cardiomyocyte specific inhibitor of WNT/CTNNB1 signalling in the heart, is lost upon cardiac remodelling, and accompanied by aberrantly active WNT/CTNNB1 resulting in heart failure progression. We investigated KLF15 expression dynamics employing CRISPR/Cas9-based tools in mouse cardiomyocytes in vivo and in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) under the hypothesis that re-establishment of KLF15 levels in myocardial stress conditions prevents heart failure progression. Using a mouse model expressing enzymatically inactive Cas9 (dCas9) fused to transcriptional activators (VPR) under Myh6 -promoter control, we activated Klf15 in a murine pressure overload model by transverse aortic constriction. Delivery of Klf15 gRNAs targeted to the Klf15 promoter region via AAV9 induced Klf15 expression sufficiently to re-normalize Klf15 expression to transcript levels comparable to sham surgery hearts. This was accompanied by reduced decrease of fractional shortening as well as reduced cardiomyocyte hypertrophy in stressed Klf15 re-activated hearts compared to non-trageted (NT) gRNA hearts (n=3-8 per group, echo data from 4 and 8 weeks post-surgery). We achieved titratable KLF15 activation in dCas9VPR transgenic hiPSC-CM by selection of single and multiple gRNAs (n=3-4 replicates) and used these cells to generate human engineered myocardium by combining hiPSC-CM and fibroblasts which we subjected to isometric contractions in order to induce mechanical stress, which resulted in KLF15 expressional decrease in line with our in vivo data. This transcriptional loss was rescued in CRISPR/dCas9VPR hiPSC-CM targeted to the KLF15 locus compared to controls (n=6-9/2/4 tissues per group/casting sessions/differentiations). Additionally, TGFB1 induced cardiomyocyte stress resulted in decreased KLF15 expression levels in 2D hiPSC-CM cultures which were rescued by dCas9VPR- KLF15 targeting (n=3 experiments). In conclusion, we report controllable gene activity by CRISPR/dCas9VPR to restore the loss of KLF15 in stressed mouse and human cardiomyocytes. We furthermore evaluate the potential to gain full control over gene dose titratability with these models to validate and define novel therapeutic targets for the prevention of heart failure progression.

Physical exercise prevents age-related heart dysfunction induced by high-salt intake and heart salt-specific overexpression in Drosophila.

A long-term high-salt intake (HSI) seems to accelerate cardiac aging and age-related diseases, but the molecular mechanism is still not entirely clear. Exercise is an effective way to delay cardiac aging. However, it remains unclear whether long-term exercise (LTE) can protect heart from aging induced by high-salt stress. In this study, heart CG2196(salt) specific overexpression (HSSO) and RNAi (HSSR) was constructed by using the UAS/hand-Gal4 system in Drosophila. Flies were given exercise and a high-salt diet intervention from 1 to 5 weeks of age. Results showed that HSSR and LTE remarkably prevented heart from accelerated age-related defects caused by HSI and HSSO, and these defects included a marked increase in heart period, arrhythmia index, malondialdehyde (MDA) level, salt expression, and dTOR expression, and a marked decrease in fractional shortening, SOD activity level, dFOXO expression, PGC-1α expression, and the number of mitochondria and myofibrils. The combination of HSSR and LTE could better protect the aging heart from the damage of HSI. Therefore, current evidences suggested that LTE resisted HSI-induced heart presenility via blocking CG2196(salt)/TOR/oxidative stress and activating dFOXO/PGC-1α. LTE also reversed heart presenility induced by cardiac-salt overexpression via activating dFOXO/PGC-1α and blocking TOR/oxidative stress.

B-PO04-006 CARDIAC SPECIFIC TSPO GENE SILENCING IMPROVES ELECTRICAL REMODELING AND POST-ISCHEMIC ARRHYTHMIAS IN HYPERTROPHIED HEARTS

Mice with cardiac specific conditional knockout of TSPO were found to be protected against mechanical dysfunction in a murine model of left ventricular hypertrophy (LVH) secondary to transaortic constriction (TAC). The role of TSPO in LVH related electrophysiological (EP) remodeling and arrhythmia remains unknown. We sought to determine the impact of cardiotropic AAV-mediated gene silencing of TSPO following onset of LVH as a therapeutic tool for reversing pre-established pathological remodeling and arrhythmia propensity. 8-wk old male mice underwent TAC and were monitored weekly by echocardiography. 3 wks following TAC, mice were randomized to receive AAV mediated gene transfer of short hairpin shRNA against TSPO (shTSPO, N=6, 5E10vg) or control (Ctrl, N =7). 6-wks following TAC, mice were sacrificed for detailed EP measurements using optical action potential mapping. Myocyte TSPO knockdown (35%, p=0.03) using shTSPO failed to reverse TAC-induced decrease in fractional shortening. shTSPO, however, prevented the increase in HW/BW ratio caused by TAC and significantly improved EP properties by blunting rate-dependent myocardial conduction slowing relative to Ctrl (A). Moreover, shTSPO suppressed post-ischemic episodes of VF in TAC hearts (0/4 shTSPO vs 2/5 Ctrl) likely by preventing APD overshoot during reperfusion (B). TAC-related VF in Ctrl but not shTSPO hearts was supported by focal activation and intermittent meandering rotors. Our findings reveal a potential role for myocardial TSPO gene silencing following onset of remodeling in improving EP properties and suppressing post-ischemic arrhythmias in LVH.

An antibody against L1 cell adhesion molecule inhibits cardiotoxicity by regulating persistent DNA damage

Targeting the molecular pathways underlying the cardiotoxicity associated with thoracic irradiation and doxorubicin (Dox) could reduce the morbidity and mortality associated with these anticancer treatments. Here, we find that vascular endothelial cells (ECs) with persistent DNA damage induced by irradiation and Dox treatment exhibit a fibrotic phenotype (endothelial–mesenchymal transition, EndMT) correlating with the colocalization of L1CAM and persistent DNA damage foci. We demonstrate that treatment with the anti-L1CAM antibody Ab417 decreases L1CAM overexpression and nuclear translocation and persistent DNA damage foci. We show that in whole-heart–irradiated mice, EC-specific p53 deletion increases vascular fibrosis and the colocalization of L1CAM and DNA damage foci, while Ab417 attenuates these effects. We also demonstrate that Ab417 prevents cardiac dysfunction-related decrease in fractional shortening and prolongs survival after whole-heart irradiation or Dox treatment. We show that cardiomyopathy patient-derived cardiovascular ECs with persistent DNA damage show upregulated L1CAM and EndMT, indicating clinical applicability of Ab417. We conclude that controlling vascular DNA damage by inhibiting nuclear L1CAM translocation might effectively prevent anticancer therapy-associated cardiotoxicity.

Cardiomyocyte‐Specific Transgenic MMP9 Overexpression Induces Cardiac Remodeling

Objective Activated matrix metalloproteinase-9 (MMP9), a collagenase, promotes cardiac remodeling leading to dysfunction. MMP9 is secreted by a wide variety of cells including cardiomyocytes. Although the role of MMP9 secreted from leukocytes and fibroblasts have been studied, very little is known about the role of cardiomyocyte-specific MMP9. Hypothesis Cardiomyocyte-specific MMP9 overexpression induces cardiac remodeling. Materials and Methods We have developed a novel cardiomyocytes-specific MMP9 transgenic (M9Tg) mouse strain. We used alpha-myosin heavy chain promoter and knocked in MMP9 gene using CRISPR-Cas9 method. After validation through genotyping, we performed gravimetric analyses and evaluated cardiac function by M-Mode echocardiography (short-axis), and hemodynamic changes by pressure-volume loop (open-chest, 4mm catheter, Transonic) studies. Results Eighteen-week old male M9Tg mice had a higher body weight and other organ's weight compared to the sibling WT. In four mice, the comparison of M9Tg/WT: body weight (g) = 26.8/24.8, heart weight (mg) = 119.0/107.0, Liver (mg) = 1309.5/1203.3, Kidney (mg) = 159.6/141.6, lung (mg) = 154.8/141.3). There was no significant change in the size of these organs relative to their body weight in M9Tg mice. The M-mode echocardiography analysis of three mice showed a decrease in cardiac output (WT= 15.8 ± 1.1, M9Tg =13.5 ± 0.8, P= 0.038) and a trend of decrease in fractional shortening (WT =27.4 ± 3.6, M9Tg = 24.9 ± 2.0, P = 0.231) and an increase in left ventricular mass (WT = 75.1 ± 4.1, M9Tg = 80.5 ± 3.8, P = 0.12). The pressure-volume loop study with two WT and three M9Tg mice showed an increase in dp/dt max (average, WT =5881.5, M9Tg = 7211.3 mmHg/s) and decrease in end systolic volume (WT = 22.1, M9Tg = 17.4 µL), suggesting a potential increase in cardiac contractility. Conclusion Cardiomyocyte-specific overexpression of MMP9 potentially increased left ventricular mass and cardiac contractility without altering fractional shortening. Further studies are ongoing to increase the sample size and examine the changes in the heart at the histological and molecular levels.

Klotho Deficiency Causes Heart Aging via Impairing the Nrf2-GR Pathway.

Cardiac aging is an important contributing factor for heart failure, which affects a large population but remains poorly understood. The purpose of this study is to investigate whether Klotho plays a role in cardiac aging. Heart function declined in old mice (24 months), as evidenced by decreases in fractional shortening, ejection fraction, and cardiac output. Heart size and weight, cardiomyocyte size, and cardiac fibrosis were increased in old mice, indicating that aging causes cardiac hypertrophy and remodeling. Circulating Klotho levels were dramatically decreased in old mice, which prompted us to investigate whether the Klotho decline may cause heart aging. We found that Klotho gene mutation (KL-/-) largely decreased serum klotho levels and impaired heart function. Interestingly, supplement of exogenous secreted Klotho prevented heart failure, hypertrophy, and remodeling in both old mice and KL (-/-) mice. Secreted Klotho treatment inhibited excessive cardiac oxidative stress, senescence and apoptosis in old mice and KL (-/-) mice. Serum phosphate levels in KL (-/-) mice were kept in the normal range, suggesting that Klotho deficiency-induced heart aging is independent of phosphate metabolism. Mechanistically, Klotho deficiency suppressed GR (glutathione reductase) expression and activity in the heart via inhibition of transcription factor Nrf2 (nuclear factor-erythroid 2 p45-related factor 2). Furthermore, cardiac-specific overexpression of GR prevented excessive oxidative stress, apoptosis, and heart failure in both old and KL (-/-) mice. Klotho deficiency causes cardiac aging via impairing the Nrf2-GR pathway. Supplement of exogenous secreted Klotho represents a promising therapeutic strategy for aging-associated cardiomyopathy and heart failure.

Abstract 16343: Sarpogrelate, a Selective Serotonin 2a Receptor Antagonist, Suppresses Pressure Overlaod-induced Cardiac Hypertrophy and Systolic Dysfunction Through Inhibiting Erk1/2/gata4 Pathway

Introduction: Serotonin (5-HT), a neurohormone involved in a wide range of physiological functions, has generated much interest in recent years regarding its potential role in cardiac function. It is reported that sarpogrelate, a selective 5-HT2A receptor antagonist, possesses cardioprotective effect against myocardial infarction, however, the precise molecular mechanism of the effect is still unclear. In this study, we examined the effect of sarpogrelate on pressure overload-induced development of heart failure, another heart failure model. Methods: First, primary cultured cardiomyocytes were treated with 1 μM sarpogrelate and then stimulated with various hypertrophic stimuli. Cardiomyocytes were stained with anti-actinin antibody and the surface area of the cells were measured. The phosphorylation levels of ERK1/2 and GATA4 were assessed by western blotting. Next, C57BL/6j male mice were subjected to a transverse aortic constriction (TAC) and sham operation. One day after the operation, the mice were randomly divided into 3 groups: sarpogrelate at 1 mg/kg or 5 mg/kg, and vehicle as a control. Daily oral administration was repeated for 8 weeks. Results: Sarpogrelate significantly suppressed an increase in the surface area of cardiomyocytes induced not only by 5-HT, but also by phenylephrine, angiotensin II and ET-1. Sarpogrelate suppressed phenylephrine-induced phosphorylation of ERK1/2 and GATA4. In a mice model of heart failure, echocardiographic analysis showed that 5 mg/kg sarpogrelate significantly prevented a TAC-induced increase in posterior left ventricular wall thickness and a decrease in fractional shortening at 8 weeks after the operation. One mg/kg sarpogrelate also suppressed TAC-induced increase in HW/BW ratio, myocardial cell diameter and the mRNA levels of ANF and BNP. Moreover, 1 mg/kg sarpogrelate significantly suppressed TAC-induced phosphorylation of ERK1/2. Conclusions: These results indicate that sarpogrelate significantly suppresses cardiomyocyte hypertrophy and the development of heart failure via at least, in part, by inhibition of ERK1/2/GATA4 pathway. These findings suggest that sarpogrelate may be an effective agent for heart failure therapy.