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BACKGROUND: The most common cause of chronic heart failure in the US is secondary or primary dilated cardiomyopathy (DCM). The DCM phenotype exhibits changes in the expression of genes that regulate contractile function and pathologic hypertrophy. However, it is unclear if any of these alterations in gene expression are disease producing or modifying. MATERIALS AND METHODS: One approach to providing evidence for cause-effect of a disease-influencing gene is to quantitatively compare changes in phenotype to changes in gene expression by employing serial measurements in a longitudinal experimental design. We investigated the quantitative relationships between changes in gene expression and phenotype n 47 patients with idiopathic DCM. In endomyocardial biopsies at baseline and 6 months later, we measured mRNA expression of genes regulating contractile function (beta-adrenergic receptors, sarcoplasmic reticulum Ca(2) + ATPase, and alpha- and beta-myosin heavy chain isoforms) or associated with pathologic hypertrophy (beta-myosin heavy chain and atrial natriuretic peptide), plus beta-adrenergic receptor protein expression. Left ventricular phenotype was assessed by radionuclide ejection fraction. RESULTS: Improvement in DCM phenotype was directly related to a coordinate increase in alpha- and a decrease in beta-myosin heavy chain mRNA expression. In contrast, modification of phenotype was unrelated to changes in the expression of beta(1)- or beta(2)-adrenergic receptor mRNA or protein, or to the mRNA expression of sarcoplasmic reticulum Ca(2) + ATPase and atrial natriuretic peptide. CONCLUSION: We conclude that in human DCM, phenotypic modification is selectively associated with myosin heavy chain isoform changes. These data support the hypothesis that myosin heavy chain isoform changes contribute to disease progression in human DCM.

Using quantitative RT-PCR in RNA from right ventricular (RV) endomyocardial biopsies from intact nonfailing hearts, and subjects with moderate RV failure from primary pulmonary hypertension (PPH) or idiopathic dilated cardiomyopathy (IDC), we measured expression of genes involved in regulation of contractility or hypertrophy. Gene expression was also assessed in LV (left ventricular) and RV free wall and RV endomyocardium of hearts from end-stage IDC subjects undergoing heart transplantation or from nonfailing donors. In intact failing hearts, downregulation of b 1 -receptor mRNA and protein, upregulation of atrial natriuretic peptide mRNA expression, and increased myocyte diameter indicated similar degrees of failure and hypertrophy in the IDC and PPH phenotypes. The only molecular phenotypic difference between PPH and IDC RVs was upregulation of b 2 -receptor gene expression in PPH but not IDC. The major new findings were that ( a ) both nonfailing intact and explanted human ventricular myocardium expressed substantial amounts of a -myosin heavy chain mRNA ( a -MHC, 23‐34% of total), and ( b ) in heart failure a -MHC was downregulated (by 67‐84%) and b -MHC gene expression was upregulated. We conclude that at the mRNA level nonfailing human heart expresses substantial a -MHC. In myocardial failure this alteration in gene expression of MHC isoforms, if translated into protein expression, would decrease myosin ATPase enzyme velocity and slow speed of contraction. ( J. Clin. Invest. 1997. 100:2315‐2324.) Key words: a -myosin

Hypothesis/introduction A polymorphic marker within the angiotensin-converting enzyme (ACE) gene has been associated with circulating and tissue ACE activity and with a variety of forms of cardiovascular disease. Since angiotensin II (Ang II) causes pulmonary vasoconstriction and vascular and myocardial remodelling, we postulated a role for the renin-angiotensin system and the ACE DD genotype in the pathophysiology of primary pulmonary hypertension (PPH) and in the right ventricular response to pressure overload in these patients. Methods and results The incidence of the ACE DD genotype was evaluated in 60 patients with severe PPH compared with two normal control populations, a group of healthy population-based controls (n=158) and subjects found suitable for cardiac organ donation (n=79). Genomic DNA extracted from peripheral leukocytes was amplified using the polymerase chain reaction to detect polymorphic markers. Haemodynamics were determined by right heart catheterisation in a subset of the PPH patients. The frequency of the ACE DD genotype was 45% in the patients with PPH, compared with 24% in the organ donors, and 28% in populationbased healthy controls (p=0.01 for chi-square test). Of the 32 PPH patients with baseline haemodynamics, 12 exhibited the ACE DD genotype and 20 were non-DD. While the mean pulmonary artery pressure and the duration of symptoms attributable to pulmonary hypertension was not different between the DD and non-DD groups, cardiac output was significantly lower (3.29±0.27 vs. 5.07±0.37 L/minute, p=0.002) and the trouvemean right atrial pressure tended to be higher (8.85±1.29 vs. 4.92±1.27 mmHg, p=0.08) in the non-DD group. The reduction in cardiac output seen in the non-DD group was not due to a difference in heart rate, but to a significant reduction in stroke volume, consistent with a decreased contractile state. In addition, non-DD patients exhibited a significantly worse functional capacity (NYHA Class 3.14±0.12 vs. 2.40±0.28, p=0.02). Conclusions 1) The ACE DD genotype is significantly increased in patients with severe PPH compared with normal controls, suggesting that certain individuals may be genetically predisposed to developing pulmonary hypertension. 2) The ACE DD genotype is associated with preserved right ventricular function in PPH patients, supporting a compensatory myocardial or inotropic role for Ang II in the pressure overloaded right ventricle.

The beta(1)-adrenergic receptor (AR) is the dominant subtype in non-failing and failing myocardium. beta(1)-AR signaling, by the endogenous neurotransmitter norepinephrine, is central to the regulation of myocardial contractility. In heart failure, the beta(1)-AR undergoes subtype-selective downregulation which may protect against the increased cardiac adrenergic drive associated with this pathophysiological state. To examine the hypothesis that chronically increased beta(1)-AR mediated signaling has adverse myocardial effects, transgenic mice overexpressing the human beta(1)-AR in a cardiac-selective context were produced, utilizing an alpha-myosin heavy chain (MHC) promoter. In these mice, beta(1)-AR protein abundance was approximately 24-46-fold (1-2 pmol/mg protein) that of wild-type mice. Histopathological examination of young (4 months old) and old (approximately 9 months old) transgenic mouse hearts consistently demonstrated large areas of interstitial replacement fibrosis, marked myocyte hypertrophy and myofibrilar disarray. In addition, increased expression of the pre-apoptotic marker, Bax, was observed coincident with regions of fibrosis accompanied by an increased apoptotic index, as measured by TUNEL assay. Older non-transgenic mice exhibited a slight tendency towards a decreased fractional shortening, whereas older beta(1)-AR transgenic mice had a marked reduction in fractional shortening (%FS approximately 30) as determined by echocardiography. Additionally, older beta(1)-AR transgenic mice had an increased left ventricular chamber size. In summary, cardiac-directed overexpression of the human beta(1)-AR in transgenic mice leads to a significant histopathological phenotype with no apparent functional consequence in younger mice and a variable degree of cardiac dysfunction in older animals. This model system may ultimately prove useful for investigating the biological basis of adrenergically-mediated myocardial damage in humans.

Background —Increased expression of Ca 2+ -sensitive protein kinase C (PKC) isoforms may be important markers of heart failure. Our aim was to determine the relative expression of PKC-β1, -β2, and -α in failed and nonfailed myocardium. Methods and Results —Explanted hearts of patients in whom dilated cardiomyopathy or ischemic cardiomyopathy was diagnosed were examined for PKC isoform content by Western blot, immunohistochemistry, enzymatic activity, and in situ hybridization and compared with nonfailed left ventricle. Quantitative immunoblotting revealed significant increases of >40% in PKC-β1 ( P P P −1 · min −1 , P −1 · mg −1 (versus 42.5 pmol · min −1 · mg −1 in nonfailed, P Conclusions —In failed human heart, PKC-β1 and -β2 expression and contribution to total PKC activity are significantly increased. This may signal a role for Ca 2+ -sensitive PKC isoforms in cardiac mechanisms involved in heart failure.

Background The regulation of angiotensin II receptors and the two major subtypes (AT 1 and AT 2 ) in chronically failing human ventricular myocardium has not been previously examined. Methods and Results Angiotensin II receptors were measured by saturation binding of 125 I-[Sar 1 ,Ile 8 ]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 AT 1 and AT 2 fractions were determined by use of an AT 1 -selective antagonist, losartan. β-Adrenergic receptors were also measured by binding of 125 I-iodocyanopindolol with the β 1 and β 2 fractions determined by use of a β 1 -selective antagonist, CGP20712A. AT 1 but not AT 2 density was significantly decreased in the combined (IDC+ISC) failing left ventricles (nonfailing: AT 1 4.66±0.48, AT 2 2.73±0.39; failing: AT 1 3.20±0.29, AT 2 2.70±0.33 fmol/mg protein; mean±SE). The decrease in AT 1 density was greater in the IDC than in the ISC left ventricles (IDC: 2.73±0.40, P P =NS versus nonfailing). β 1 but not β 2 density was decreased in the failing left ventricles. AT 1 density was correlated with β 1 density in all left ventricles ( r =.43). AT 1 density was also decreased in IDC right ventricles. In situ reverse transcription–polymerase chain reaction in sections of nonfailing and failing ventricles indicated that AT 1 mRNA was present in both myocytes and nonmyocytes. Conclusions AT 1 receptors are selectively downregulated in failing human ventricles, similar to the selective downregulation of β 1 receptors. The relative lack of AT 1 downregulation in ISC hearts may be related to differences in the degree of ventricular dysfunction.

Carvedilol is an adrenoceptor antagonist which modulates the activity not only of beta 1 and beta 2 but also of alpha 1 adrenergic receptors present on the cell surface membrane of the human cardiac myocyte. In the heart, carvedilol has approximately 7 times higher potency for beta 1 and beta 2 adrenoceptors, but in the doses 50-100 mg . day-1 used in clinical practice, it is essentially non-selective. In human myocardial preparations and in cultured heart cells, carvedilol has no intrinsic sympathomimetic activity but is able to identify high affinity agonist-binding receptors whose pharmacological signature is reduction in binding by incubation with guanine nucleotides (guanine nucleotide-modulatable binding). This property is more prominent for the human beta 2 than for the beta 1 adrenoceptor. The property of gaunine nucleotide-modulatable binding for carvedilol and structurally related bucindolol correlates with their ability to directly down-regulate beta 1-like receptors present in cultured chick myocytes, and with a lack of reversal of down-regulation of cardiac beta-receptors in patients with heart failure. Carvedilol does not exhibit high levels of inverse agonist activity, which may contribute to its good tolerability in subjects with heart failure. These data indicate that carvedilol produces a high degree of adrenergic receptor blockade in the failing human heart, and does not re-sensitize the beta-receptor pathway to stimulation by adrenergic agonists.

Background The quinolinone compounds OPC-8212 (vesnarinone), OPC-18790, and OPC-8490 are members of a family of unique positive inotropic compounds that have no positive chronotropic effects. In subjects with heart failure, the prototypic compound OPC-8212 may reduce morbidity and mortality at low doses but increase mortality at high doses. Methods and Results To further characterize the inotropic mechanism(s) of action of these compounds, we investigated the effects of OPC-8490, a water-soluble quinolinone, on the inotropic response, inhibition of phosphodiesterase (PDE), and action potential in human ventricular myocardial preparations. In isolated right ventricular trabeculae and membranes prepared from left ventricular myocardium, OPC-8490 produced dose-related positive inotropic effects, inhibited type III PDE activity, and prolonged action potential. Comparative experiments with other PDE inhibitors, sodium channel agonists, and potassium channel antagonists indicated that the positive inotropic effects are due to PDE inhibition, whereas the action potential effects of OPC-8490 are due to effects on ion channels. Conclusions We conclude that OPC-8490 produces selective positive inotropic effects because of type III PDE inhibition combined with ion channel effects, with the latter property inhibiting the positive chronotropic response usually associated with agents that increase intracellular cAMP concentrations.

Background Ten percent to 20% of potential cardiac donors with brain injury and no previous cardiac history have myocardial dysfunction. We assessed components of the β-receptor–G-protein–adenylyl cyclase complex as well as the contractile response in 10 explanted acutely failing human hearts (donor heart dysfunction [DHD]) and compared the results with 13 age-matched nonfailing (NF) organ donor controls. Methods and Results As measured by echocardiography, all DHD hearts exhibited a decreased shortening fraction (16±2%, mean±SEM). Although total and subpopulation β-receptor densities measured by [ 125 I]iodocyanopindolol (ICYP) were similar in the DHD and NF groups, DHD hearts exhibited a 30% decrease in maximum isoproterenol-stimulated adenylyl cyclase activity and a 50% decrease in the maximal response to zinterol. DHD hearts also exhibited decreases in adenylyl cyclase maximal stimulation by forskolin (211±25 [DHD] versus 295±23 [NF] pmol cAMP · min −1 · mg −1 , P −1 · mg −1 , P 2+ , a direct activator of adenylyl cyclase. Right ventricular trabeculae removed from DHD hearts exhibited a profound decrease in the contractile response to isoproterenol (8.7±1 [DHD] versus 22±2 [NF] mN, P P =.03). Morphological examination of two hearts revealed some ultrastructural evidence suggestive of catecholamine-mediated injury, but there was no difference in tissue creatine kinase activity between the two groups. Conclusions Compared with NF hearts, DHD hearts exhibit marked uncoupling of β 1 - and β 2 -adrenergic receptors from adenylyl cyclase and contractile response stimulation as well as decreased intrinsic systolic function. Thus, acute myocardial dysfunction accompanying brain injury is characterized by marked alterations in β-adrenergic signal transduction as well as changes in the contractile apparatus, and this profile is markedly different from what occurs in the chronically failing human heart.

It has been proposed that the contribution of myocardial tissue angiotensin converting enzyme (ACE) to angiotensin II (Ang II) formation in the human heart is low compared with non-ACE pathways. However, little is known about the actual in vivo contribution of these pathways to Ang II formation in the human heart. To examine angiotensin II formation in the intact human heart, we administered intracoronary 123I-labeled angiotensin I (Ang I) with and without intracoronary enalaprilat to orthotopic heart transplant recipients. The fractional conversion of Ang I to Ang II, calculated after separation of angiotensin peptides by HPLC, was 0.415 +/- 0.104 (n = 5, mean +/- SD). Enalaprilat reduced fractional conversion by 89%, to a value of 0.044 +/- 0.053 (n = 4, P = 0.002). In a separate study of explanted hearts, a newly developed in vitro Ang II-forming assay was used to examine cardiac tissue ACE activity independent of circulating components. ACE activity in solubilized left ventricular membrane preparations from failing hearts was 49.6 +/- 5.3 fmol 125I-Ang II formed per minute per milligram of protein (n = 8, +/- SE), and 35.9 +/- 4.8 fmol/min/mg from nonfailing human hearts (n = 7, P = 0.08). In the presence of 1 microM enalaprilat, ACE activity was reduced by 85%, to 7.3 +/- 1.4 fmol/min/mg in the failing group and to 4.6 +/- 1.3 fmol/min/mg in the nonfailing group (P < 0.001). We conclude that the predominant pathway for angiotensin II formation in the human heart is through ACE.

Carvedilol, a new beta-blocker with vasodilating properties due to alpha 1-blockade, was investigated in preparations of human ventricular myocardium. Carvedilol demonstrated a high affinity and is a slightly beta 1-selective competitive beta-blocking agent, with a KD for beta 1-receptors of approximately 4-5 nM and a mild selectivity for beta 1 vs. beta 2 receptors of 6- to 39-fold, depending on the method employed to assess subtype potency. In addition, carvedilol was also 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 in human myocardial membranes containing both beta 1- and beta 2-receptors carvedilol exhibited the unique property of guanine nucleotide modulatable binding. Despite this, no intrinsic sympathomimetic activity of carvedilol was detected in preparations of isolated human heart or in myocardial membranes. Vasodilation related to alpha 1-blockade and the lack of intrinsic activity should translate into improved tolerability and good efficacy in the treatment of heart failure.

It has been proposed that the contribution of myocardial tissue angiotensin converting enzyme (ACE) to angiotensin II (Ang II) formation in the heart is low compared to non-ACE pathways. However, little is known about the actual in vivo contribution of these pathways to Ang II formation in the human heart. Accordingly, we developed a method to quantify ACE and non-ACEmediated conversion of angiotensin I (Ang I) to Ang II across the myocardial circulation in human subjects. Using an intracoronary(lC) infusion of 123|-Ang I before and during an IC infusion of enalaprilat, we measured the fractional conversion of Ang I to Ang II in coronary sinus blood samples by taking the ratio of the labeled Ang II activity to the sum of activity corresponding to labeled Ang I and Ang II after peptide extraction and separation by HPLC (activity ratio method). Results obtained from this method were confirmed by combined HPLC/RIA of both labeled and unlabeled Ang I and Ang II (RIA method). The initial experience has been obtained in orthotopic heart transplant recipients with normal cardiac function and coronary anatomy. After a six minute IC infusion of 123|Ang I, fractional conversion of Ang I to Ang II was 0.44 ± 0.141, and 0.43 ± 0.084 (n = 4, mean ± SD) as determined, respectively, by the activity ratio and RIA methods. IC enalaprilat (0.01 mg/min for five minutes) reduced fractional conversion to 0.046 ± 0.065 (activity ratio method) or 0.033 ± 0.012 (RIA method) (n = 3, P = 0.014). Download : Download high-res image (61KB) Download : Download full-size image Conclusions 1) Ang II formation can be measured in the intact human heart; 2) ACE mediates 90% of Ang I to Ang II conversion across the myocardial circulation.

The renin-angiotensin and adrenergic nervous systems exhibit multiple levels of cross-regulation in heart failure. These systems are bidirectionally activated in concert; i.e. activation of one system activates the other. We compared the behavior of angiotensin II AT1 and AT2 receptors with β1, -and, β2-adrenergic receptors in a high-yield crude membrane fraction prepared from nonfailing and failing human ventricular myocardium. Ang II receptors were measured by 125I saralasin binding, with Bmax determined by saturation binding displaceable by 1 μM cold saralasin. AT1 and AT2 receptor fractions were determined by the amount of specific binding displaceable by 1 μM losartan. β1-adrenergic receptor density was determined by saturation binding of 125I ICYP, with the, β1 fraction determined by binding displaceable by 0.2 μM CGP 20712A. Results in end-stage human left ventricular myocardium failing as a result of idiopathic dilated cardiomyopathy were compared to nonfailing controls taken from age- and gender-matched organ donors not used for transplant because of blood type or body size mismatch: (Receptor density is in fmol/mg ± SEM) β1-AR β2-AR AT1 AT2 Nonfailing (n = 6) 59.0 ± 9.4 20.7 ± 4.0 4.14 ± 0.62 1.52 ± 0.43 Failing (n = 6) 28.3 ± 2.8 * 17.2 ± 2.6 1.53 ± 0.57 * 2.68 ± 0.51 * p l 0.05 The down-regulation of β1 AR and AT1 receptors was significantly related (r = 0.62, n = 12, p l 0.05) Conclusions (1) Compared to β adrenergic receptors ang II receptors are very low density in the human heart. (2) The AT1 receptor sUbtype predominates in the nonfailing human heart. (3) ATl but not AT2 receptors are downregulated in failing heart. (4) Down-regulation of Ang-II AT1 receptor is similar in degree to down-regulation of β1-adrenergic receptors. These data suggest that the AT1 and β1 receptors are respectively exposed to increased concentrations of mutually activatedlinduced norepinephrine and Ang-II in the failing human heart.