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Background: The phosphodiesterase 3A (PDE3A) gene encodes a PDE that regulates cardiac myocyte cyclic adenosine monophosphate (cAMP) levels and myocardial contractile function. PDE3 inhibitors (PDE3i) are used for short-term treatment of refractory heart failure (HF), but do not produce uniform long-term benefit., Objectives: The authors tested the hypothesis that drug target genetic variation could explain clinical response heterogeneity to PDE3i in HF., Methods: PDE3A promoter studies were performed in a cloned luciferase construct. In human left ventricular (LV) preparations, mRNA expression was measured by reverse transcription polymerase chain reaction, and PDE3 enzyme activity by cAMP-hydrolysis., Results: The authors identified a 29-nucleotide (nt) insertion (INS)/deletion (DEL) polymorphism in the human PDE3A gene promoter beginning 2,214 nt upstream from the PDE3A1 translation start site. Transcription factor ATF3 binds to the INS and represses cAMP-dependent promoter activity. In explanted failing LVs that were homozygous for PDE3A DEL and had been treated with PDE3i pre-cardiac transplantation, PDE3A1 mRNA abundance and microsomal PDE3 enzyme activity were increased by 1.7-fold to 1.8-fold (p < 0.05) compared with DEL homozygotes not receiving PDE3i. The basis for the selective up-regulation in PDE3A gene expression in DEL homozygotes treated with PDE3i was a cAMP response element enhancer 61 nt downstream from the INS, which was repressed by INS. The DEL homozygous genotype frequency was also enriched in patients with HF., Conclusions: A 29-nt INS/DEL polymorphism in the PDE3A promoter regulates cAMP-induced PDE3A gene expression in patients treated with PDE3i. This molecular mechanism may explain response heterogeneity to this drug class, and may inform a pharmacogenetic strategy for a more effective use of PDE3i in HF., (Copyright © 2019 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Myocardial H 2 receptor activation contributes to heart failure (HF) in preclinical models, and H 2 receptor antagonists are associated with decreased HF incidence. This study evaluated whether H 2 histamine receptor (HRH2) single nucleotide polymorphisms (SNPs) are associated with HF incidence and whether myocardial transcript abundance is associated with HF recovery. The association of SNPs in HRH2 with incident HF was characterized using Cox proportional hazards regression among participants in the Multi-Ethnic Study of Atherosclerosis. Differences in myocardial HRH2 transcripts were characterized in participants with dilated cardiomyopathy comparing 6 "super-responders" with 6 nonresponders to β blockade in the Beta-Blocker Effect on Remodeling and Gene Expression Trial. In MESA, no candidate SNP was associated with HF in black, Hispanic, or white participants. The rs2241562 minor allele was present only in Chinese participants and the adjusted HF hazard among those with 1 or more copies of this allele was 3.7, 95% confidence interval 1.0 to 13.4. In BORG, super-responders to β blockade had higher levels of myocardial HRH2 transcript at baseline compared with nonresponders (fragments per kilobase per transcript per million mapped reads: Variant 2, 5.5 ± 1.1 compared with 3.2 ± 0.8 in nonresponders, p = 0.002; Variant 1 + 2, 32.1 ± 7.4 compared with 23.3 ± 4.2 in nonresponders, p = 0.04). In conclusion, the presence of a minor allele at rs2241562 was associated with increased HF incidence in Chinese participants. Differences in myocardial HRH2 transcript abundance were seen in participants with dilated cardiomyopathy who responded to β blockade. These observations support the hypothesis that HRH2 is involved in the pathogenesis of HF., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: In dilated cardiomyopathies (DCMs) changes in expression of protein-coding genes are associated with reverse remodeling, and these changes can be regulated by microRNAs (miRs). We tested the general hypothesis that dynamic changes in myocardial miR expression are predictive of β-blocker-associated reverse remodeling., Methods: Forty-three idiopathic DCM patients (mean left ventricular ejection fraction 0.24 ± 0.09) were treated with β-blockers. Serial ventriculography and endomyocardial biopsies were performed at baseline, and after 3 and 12 months of treatment. Changes in RT-PCR (candidate miRs) or array-measured miRs were compared based on the presence (R) or absence (NR) of a reverse-remodeling response, and a miR-mRNA-function pathway analysis (PA) was performed., Results: At 3 months, 2 candidate miRs were selectively changed in Rs, decreases in miR-208a-3p and miR-591. PA revealed changes in miR-mRNA interactions predictive of decreased apoptosis and myocardial cell death. At 12 months, 5 miRs exhibited selective changes in Rs (decreases in miR-208a-3p, -208b-3p, 21-5p, and 199a-5p; increase in miR-1-3p). PA predicted decreases in apoptosis, cardiac myocyte cell death, hypertrophy, and heart failure, with increases in contractile and overall cardiac functions., Conclusions: In DCMs, myocardial miRs predict the time-dependent reverse-remodeling response to β-blocker treatment, and likely regulate the expression of remodeling-associated miRs., Trial Registration: ClinicalTrials.gov NCT01798992., Funding: NIH 2R01 HL48013, 1R01 HL71118 (Bristow, PI); sponsored research agreements from Glaxo-SmithKline and AstraZeneca (Bristow, PI); NIH P20 HL101435 (Lowes, Port multi-PD/PI); sponsored research agreement from Miragen Therapeutics (Port, PI)., Competing Interests: C.C. Sucharov, J.D. Port, and M.R. Bristow have stock (each less than 0.1% of total outstanding shares) in Miragen Therapeutics, and M.R. Bristow is member of the Miragen Scientific Advisory Board.

Background: With increasing age, human ventricular myocardium exhibits selective downregulation of β1-adrenergic receptors (β1-ARs). We tested the hypothesis that sex differences exist in age-related changes in β1-ARs., Methods: Left (LV) and right (RV) ventricular tissue was obtained from 61 unplaceable potential organ donor hearts ages 1 to 71 years with no known cardiac history and from LVs removed from 56 transplant recipients with idiopathic dilated cardiomyopathy. β1-AR and β2-AR densities, the frequency of β1-AR389 gene variants, and β-AR function were determined., Results: Sex had a marked effect on the age-related decrease in β1-ARs. Female LVs had more pronounced downregulation (by 42% [p < 0.001] vs 22% [p = 0.21] in 31 male LVs) comparing the youngest (average age, 15.3 ± 5.5 years) to the oldest (average age, 50.8 ± 9.1 years) sub-groups. On regression analyses, female LVs exhibited a closer relationship between β1-AR density and age (r = -0.78, p <0.001 vs r = -0.46, p = 0.009 in males), with a second-degree polynomial yielding the best fit. There was no statistically significant relationship of β1-ARs to age in female or male idiopathic dilated cardiomyopathy LVs., Conclusions: Sex affects age-related β-AR downregulation in normal human ventricles, with females exhibiting more profound decreases with increasing age. The curvilinear relationship between age and receptor density that plateaus around age 40 in women suggests an effect of sex hormones on β1-AR expression in the human heart., (Copyright © 2016 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: When β-blockers produce reverse-remodeling in idiopathic dilated cardiomyopathy, they partially reverse changes in fetal-adult/contractile protein, natriuretic peptide, SR-Ca(2+)-ATPase gene program constituents. The objective of the current study was to further test the hypothesis that reverse-remodeling is associated with favorable changes in myocardial gene expression by measuring additional contractile, signaling, and metabolic genes that exhibit a fetal/adult expression predominance, are thyroid hormone-responsive, and are regulated by β1-adrenergic receptor signaling. A secondary objective was to identify which of these putative regulatory networks is most closely associated with observed changes., Methods and Results: Forty-seven patients with idiopathic dilated cardiomyopathy (left ventricular ejection fraction, 0.24±0.09) were randomized to the adrenergic-receptor blockers metoprolol (β1-selective), metoprolol+doxazosin (β1/α1), or carvedilol (β1/β2/α1). Serial radionuclide ventriculography and endomyocardial biopsies were performed at baseline, 3, and 12 months. Expression of 50 mRNA gene products was measured by quantitative polymerase chain reaction. Thirty-one patients achieved left ventricular ejection fraction reverse-remodeling response defined as improvement by ≥0.08 at 12 months or by ≥0.05 at 3 months (Δ left ventricular ejection fraction, 0.21±0.10). Changes in gene expression in responders versus nonresponders were decreases in NPPA and NPPB and increases in MYH6, ATP2A2, PLN, RYR2, ADRA1A, ADRB1, MYL3, PDFKM, PDHX, and CPT1B. All except PDHX involved increase in adult or decrease in fetal cardiac genes, but 100% were concordant with changes predicted by inhibition of β1-adrenergic signaling., Conclusions: In addition to known gene expression changes, additional calcium-handling, sarcomeric, adrenergic signaling, and metabolic genes were associated with reverse-remodeling. The pattern suggests a fetal-adult paradigm but may be because of reversal of gene expression controlled by a β1-adrenergic receptor gene network., Clinical Trial Registration: URL: www.clinicaltrials.gov. Unique Identifier: NCT01798992., (© 2015 American Heart Association, Inc.)

Background: Heart failure is associated with reversal to a fetal gene expression pattern of contractile and metabolic genes. Substantial recovery of ventricular function with assist devices is rare. Our goal was to evaluate the effects of assist devices on fetal gene expression and hypoxia inducible factor-1 alpha (HIF-1 alpha), a transcriptional factor in hypoxic signaling., Methods: Human heart tissue was obtained from the left ventricular apex at the time of assist device implantation and again from the left ventricular free wall during cardiac transplantation. Non-failing tissue was obtained from unused hearts from human donors. Gene expression was measured with the Affymetrix 133 plus 2 Array. HIF-1 alpha was measured by Western blotting with commercially available antibodies., Results: Heart failure was associated with a decrease in alpha-myosin heavy chain and sarcoplasmic reticulum-Ca(2+) adenosine triphosphatase messenger RNA expression along with an increase in skeletal tropomyosin. This pattern persisted after assist device therapy. Heart failure was also associated with abnormalities in regulatory metabolic genes including glucose transporter 1 (GLUT1). These patterns also persisted after assist device therapy despite a reduction in atrial natriuretic peptide expression and normalization of HIF-1 alpha., Conclusions: Failure of assist devices to produce sustained recovery of myocardial contractile function may be due in part to persistent fetal transcriptional patterns of contractile and metabolic genes.

The calcium/calmodulin-dependent phosphatase calcineurin plays a central role in the control of cardiomyocyte hypertrophy in response to pathological stimuli. Although calcineurin is present at high levels in normal heart, its activity appears to be unaffected by calcium during the course of a cardiac cycle. The mechanism(s) whereby calcineurin is selectively activated by calcium under pathological conditions has remained unclear. Here, we demonstrate that diverse signals for cardiac hypertrophy stimulate expression of canonical transient receptor potential (TRPC) channels. TRPC consists of a family of seven membrane-spanning nonselective cation channels that have been implicated in the nonvoltage-gated influx of calcium in response to G protein-coupled receptor signaling, receptor tyrosine kinase signaling, and depletion of internal calcium stores. TRPC3 expression is up-regulated in multiple rodent models of pathological cardiac hypertrophy, whereas TRPC5 expression is induced in failing human heart. We demonstrate that TRPC promotes cardiomyocyte hypertrophy through activation of calcineurin and its downstream effector, the nuclear factor of activated T cells transcription factor. These results define a novel role for TRPC channels in the control of cardiac growth, and suggest that a TRPC-derived pool of calcium contributes to selective activation of calcineurin in diseased heart.

Introduction: Hypoplastic left heart syndrome (HLHS) is the term used to describe a group of congenital malformations characterized by marked underdevelopment of the left side of the heart. HLHS accounts for nearly 25% of cardiac deaths in the first year of life. Although much has been reported regarding diagnosis, gross morphology and surgical treatment, no information on gene expression in HLHS myocytes is available., Methods: We examined heart tissue from patients with HLHS using routine histology, immunohistochemistry, quantitative polymerase chain reaction (PCR), two-dimensional (2-D) gel electrophoresis and protein identification by mass spectrometry., Results: Histologic examination of right and left ventricles from HLHS patients revealed characteristic features of myocyte differentiation, including striations and intercalated disc formation. Immunohistochemical staining using antibody to N-cadherin demonstrated clear development of intercalated discs between myocytes. However, many of the myocytes contained scant cytoplasm and were grouped in small, disorganized bundles separated by abundant connective tissue and dilated, thin-walled vessels. Quantitative PCR analysis demonstrated that both left and right ventricular tissue from HLHS hearts expressed the fetal or "heart failure" gene expression pattern. Two-dimensional gel electrophoresis and protein identification by mass spectrometry also confirmed that myocytes from HLHS ventricles were differentiated but expressed the fetal isoform of some cardiac specific proteins. However, HLHS myocytes in all of the heart samples (n=21) were inappropriately expressing platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31), a member of the cell adhesion molecule (CAM) family that has a primary role in the regulation of tissue morphogenesis. These findings indicate that myocytes from HLHS syndrome patients, while differentiated, have a unique gene expression pattern.

In the heart, the relative proportions of the 2 forms of the motor protein myosin heavy chain (MyHC) have been shown to be affected by a wide variety of pathological and physiological stimuli. Hearts that express the faster MyHC motor protein, alpha, produce more power than those expressing the slower MyHC motor protein, beta, leading to the hypothesis that MyHC isoforms play a major role in the determination of cardiac contractility. We showed previously that a significant amount of alphaMyHC mRNA is expressed in nonfailing human ventricular myocardium and that alphaMyHC mRNA expression is decreased 15-fold in end-stage failing left ventricles. In the present study, we determined the MyHC protein isoform content of human heart samples of known MyHC mRNA composition. We demonstrate that alphaMyHC protein was easily detectable in 12 nonfailing hearts. alphaMyHC protein represented 7.2+/-3.2% of total MyHC protein (compared with approximately 35% of the MyHC mRNA), suggesting that translational regulation may be operative; in contrast, there was effectively no detectable alphaMyHC protein in the left ventricles of 10 end-stage failing human hearts.

Background: The regulation of angiotensin II receptors and the two major subtypes (AT1 and AT2) in chronically failing human ventricular myocardium has not been previously examined., Methods and Results: Angiotensin II receptors were measured by saturation binding of 125I-[Sar1,Ile8]angiotensin II in crude membranes from nonfailing (n = 19) and failing human left ventricles with idiopathic dilated cardiomyopathy (IDC; n = 31) or ischemic cardiomyopathy (ISC; n = 21) and membranes from a limited number of right ventricles in each category. The AT1 and AT2 fractions were determined by use of an AT1-selective antagonist, losartan. beta-Adrenergic receptors were also measured by binding of 125I-iodocyanopindolol with the beta 1 and beta 2 fractions determined by use of a beta 1-selective antagonist, CGP20712A, AT1 but not AT2 density was significantly decreased in the combined (IDC + ISC) failing left ventricles (nonfailing: AT1 4.66 +/- 0.48, AT2 2.73 +/- 0.39; failing: AT1 3.20 +/- 0.29, AT2 2.70 +/- 0.33 fmol/mg protein; mean +/- SE). The decrease in AT1 density was greater in the IDC than in the ISC left ventricles (IDC: 2.73 +/- 0.40, P < .01; ISC: 3.89 +/- 0.39 fmol/mg protein, P = NS versus nonfailing). beta 1 but not beta 2 density was decreased in the failing left ventricles. AT1 density was correlated with beta 1 density in all left ventricles (r = .43). AT1 density was also decreased in IDC right ventricles. In situ reverse transcription-polymerase chain reaction in sections of nonfailing and failing ventricles indicated that AT1 mRNA was present in both myocytes and nonmyocytes., Conclusions: AT1 receptors are selectively downregulated in failing human ventricles, similar to the selective downregulation of beta 1 receptors. The relative lack of AT1 downregulation in ISC hearts may be related to differences in the degree of ventricular dysfunction.

The beta-blocker and vasodilator carvedilol was examined in preparations of human ventricular myocardium. Carvedilol is a high-affinity, slightly beta 1-selective competitive beta-blocking agent, with a KD for beta 1-receptors of approximately 4-5 nM and a selectivity of sixfold to 39-fold for beta 1-receptors rather than beta 2-receptors, depending on the method used to assess subtype potency. Carvedilol also is a potent alpha 1-blocking agent, with a beta 1: alpha 1-blocking relative potency of 1.7-fold. In human lymphocytes containing beta 2-receptors and human myocardial membranes containing both beta 1- and beta 2-receptors, carvedilol exhibited the unique property of guanine nucleotide-modulatable binding. This is a property shared with bucindolol, another beta-blocker and vasodilator that is structurally similar to carvedilol. Despite the presence of guanine nucleotide-modulatable binding, no intrinsic activity of carvedilol was detected in preparations of isolated human heart or in myocardial membranes.

Studies in myopathic hamsters have described an increase in calcium antagonist binding sites, which is presumably associated with an increase in the number of calcium channels. Such an abnormality might predispose the heart to further myocardial damage from calcium overload. We tested the hypothesis that calcium antagonist binding sites are increased in human idiopathic dilated cardiomyopathy by examining [3H]PN 200-110 and [3H]nitrendipine binding in membranes prepared from nonfailing controls and severely failing ventricles with idiopathic dilated cardiomyopathy. Despite the fact that beta receptor density was decreased by 50% in failing hearts (iodocyanopindolol Bmax 84.4 +/- 8.9 fmol/mg protein in nonfailing hearts vs 42.9 +/- 3.2 fmol/mg in failing hearts, P less than 0.01), dihydropyridine calcium antagonist binding sites were not reduced significantly by heart failure. Maximum binding of [3H]PN 200-110 was 92.9 +/- 19.4 fmol/mg protein in membranes derived from failing ventricles, and 93.5 +/- 17.4 fmol/mg in membranes derived from nonfailing ventricles (P = NS); values for [3H]nitrendipine maximum binding were similar to those for [3H]PN 200-110 and also were not reduced significantly in failing ventricles. Additionally, the dissociation constants (KD) for [3H]nitrendipine and [3H]PN 200-110 were not significantly different in failing and nonfailing heart. We conclude that dihydropyridine calcium antagonist binding sites are not altered significantly in the failing human left ventricle with idiopathic dilated cardiomyopathy.

Administration of histamine to rabbits may result in myocardial damage similar to that produced by catecholamines and the anthracycline antibiotics. To explore the mechanisms involved in histamine-mediated myocardial damage, conscious New Zealand white rabbits were pretreated with H1 and H2 receptor blocking agents, alone and in combination, and then administered histamine. Coronary artery blood flow was measured with radiolabeled microspheres in rabbits that received histamine alone, and in those that received an H1 blocking agent and histamine. Rabbits that received an H1 blocking agent had a significant reduction in morphological injury which was scored as follows: grade 1, minimal or no injury; grade 2, moderate; and grade 3, severe injury (mean pathology score = 1.1 +/- 0.28 for histamine alone vs. 0.06 +/- 0.06 with H1 receptor blockade, p less than 0.05). Animals pretreated with H2 receptor blockade (mean pathology score = 1.2 +/- 0.49) were not protected against morphological injury. Coronary blood flow decreased in animals that received histamine alone: control = 2.61 +/- 0.38 vs. 1.80 +/- 0.30 ml/min/g (p less than 0.05), and in animals pretreated with H1 blockade; control = 3.29 +/- 0.34 vs. 1.91 +/- 0.28 ml/min/g (p less than 0.01). We conclude that histamine-mediated myocardial damage appears to be mediated by the H1 receptor system and that this appears to be independent of initial changes in global coronary blood flow.

[3H]-nitrendipine binding data and isolated tissue response for five calcium antagonists were evaluated in rabbit myocardium and aorta. The [3H]-nitrendipine binding site was qualitatively identical in myocardium and aorta, as the [3H]-nitrendipine KD, KIS for nicardipine and nifedipine and interactions with verapamil, D600 and diltiazem were not different in aortic and cardiac membranes prepared by similar means. In contrast, the inhibition of the Ca2+-induced contractile response in right ventricular myocardium and aortic ring segments indicated a greater than 10,000 fold selectivity of nicardipine for antagonism of vascular responses. This resulted in a different order of potency for calcium antagonist interaction with the [3H]-nitrendipine binding site in cardiac membranes (nicardipine greater than nifedipine greater than D600 greater than verapamil greater than diltiazem) as compared to antagonism of myocardial tissue response (D600 greater than verapamil greater than or equal to nifedipine greater than nicardipine greater than or equal to diltiazem). In heart the difference between the potency of nicardipine in binding experiments and tissue response approached 4 orders of magnitude. We conclude that tissue response selectivity of calcium antagonists is not explained by heterogeneity of [3H]-nitrendipine binding sites.

We tested the hypothesis that cardiac histamine release mediates subacute doxorubicin (DXR) cardiotoxicity in rabbits. New Zealand white rabbits given DXR 20 mg/kg i.v. over 30 min developed myocardial damage 24 h later that was similar to that observed in humans. In isolated heart preparations, DXR produced dose-related cardiac histamine release at DXR concentrations of 1, 5, and 25 micrograms/ml given as 1-min exposures. Prior exposure of isolated hearts to 10 microM cromolyn sodium completely prevented histamine release from 5 micrograms/ml DXR. Pretreatment of animals with cromolyn produced significant protection against DXR-mediated subacute cardiotoxicity. We conclude that the release of cardiac histamine may be involved in the pathogenesis of anthracycline cardiotoxicity.

To identify the role of the myocardial beta-adrenergic pathway in congestive heart failure, we examined beta-adrenergic-receptor density, adenylate cyclase and creatine kinase activities, muscle contraction in vitro, and myocardial contractile protein levels in the left ventricles of failing and normally functioning hearts from cardiac-transplant recipients or prospective donors. Eleven failing left ventricles had a 50 to 56 per cent reduction in beta-receptor density, a 45 per cent reduction in maximal isoproterenol-mediated adenylate cyclase stimulation, and a 54 to 73 per cent reduction in maximal isoproterenol-stimulated muscle contraction, as compared with six normally functioning ventricles (P less than 0.05 for each comparison). In contrast, cytoplasmic creatine kinase activity, adenylate cyclase activities stimulated by fluoride ion and by histamine, histamine-stimulated muscle contraction, and levels of contractile protein were not different in the two groups (P less than 0.05). We conclude that in failing human hearts a decrease in beta-receptor density leads to subsensitivity of the beta-adrenergic pathway and decreased beta-agonist-stimulated muscle contraction. Regulation of beta-adrenergic receptors may be an important variable in cardiac failure.

Weekly injections of the catecholamine depleting agent 6-hydroxydopamine (6-OHDA) were used to denervate rabbit hearts chemically. Analyses of morphology and beta-adrenergic receptor density were made at 1, 2, and 4 weeks. Changes resulting from subacute and chronic inflammatory processes were evident by light microscopy after 1 week. At that time, electron microscopy revealed marked increases in collagen, large myocytic vacuolizations in myocytes, widened gap junctions, and myofibrillar degeneration and dropout. Receptor density was marginally increased at 2 weeks but was decreased (p less than .05) at 4 weeks (maximal [3H]dihydroalprenolol (DHA) binding in fmol/mg: 69.6 +/- 5.4 in controls vs 49.2 +/- 5.1 in 6-OHDA-treated animals). Basal, isoproterenol-stimulated and F- -stimulated adenylate cyclase activities were decreased in the 6-OHDA-treated group at 4 weeks. We conclude that administration of 6-OHDA may cause severe myocardial damage, and that this process may involve loss of some functional components of the cell membrane.

We tested the hypothesis that anthracycline-induced cardiac and renal damage is mediated by vasoactive substances. A 1-minute exposure to 5 micrograms per ml. of doxorubicin (DXR, Adriamycin) produced cardiac histamine release in isolated rabbit hearts. Under conditions in which histamine uptake and metabolism were impaired, the administration of DXR, 2 mg. per kg., over 1 minute was associated with elevations in arterial histamine and catecholamines. The chronic weekly administration of DXR produced severe cardiac and renal damage. The administration of combined histaminic and adrenergic blockade with diphenhydramine, cimetidine, phentolamine, and propranolol (DCPP) pre- and immediately post-DXR resulted in near total protection against DXR-mediated cardiac damage and prevented the majority of the renal lesions. The combined administration of diphenhydramine, cimetidine, phentolamine, and propranolol did not appear to be acting by mechanisms other than blockade of vasoactive amine receptors as cardiac uptake of DXR and the DXR antitumor response were not altered by diphenhydramine, cimetidine, phentolamine, and propranolol. This study demonstrates that anthracycline-associated cardiac and renal toxicity may be mediated by vasoactive substances and that anthracycline cardiomyopathy is potentially preventable.