Ramesh C Gupta

Effects of long-term monotherapy with enalapril, metoprolol, and digoxin on the progression of left ventricular dysfunction and dilation in dogs with reduced ejection fraction.

BACKGROUNDnRecent clinical trials have suggested that therapy with angiotensin-converting enzyme inhibitors in asymptomatic patients with reduced left ventricular (LV) function can significantly reduce the incidence of congestive heart failure compared with patients receiving placebo. In the present study, we examined the effects of long-term monotherapy with enalapril, metoprolol, and digoxin on the progression of LV systolic dysfunction and LV chamber enlargement in dogs with reduced LV ejection fraction (EF).nnnMETHODS AND RESULTSnLV dysfunction was produced in 28 dogs by multiple sequential intracoronary microembolizations. Embolizations were discontinued when LVEF was 30% to 40%. Three weeks after the last embolization, dogs were randomized to 3 months of oral therapy with enalapril (10 mg twice daily, n = 7), metoprolol (25 mg twice daily, n = 7), digoxin (0.25 mg once daily, n = 7), or no treatment (control, n = 7). As expected, in untreated dogs, LVEF decreased (36 +/- 1% versus 26 +/- 1%, P < .001) and LV end-systolic volume (ESV) and end-diastolic volume (EDV) increased during the 3-month follow-up period (39 +/- 4 versus 57 +/- 6 mL, P < .001, and 61 +/- 6 versus 78 +/- 8 mL, P < .002, respectively). In dogs treated with enalapril or metoprolol, LVEF remained unchanged or increased after therapy compared with before therapy (35 +/- 1% versus 38 +/- 3% and 35 +/- 1% versus 40 +/- 3%, respectively, P < .05), whereas ESV and EDV remained essentially unchanged. In dogs treated with digoxin, EF remained unchanged but ESV and EDV increased significantly.nnnCONCLUSIONSnIn dogs with reduced LVEF, long-term therapy with enalapril or metoprolol prevents the progression of LV systolic dysfunction and LV chamber dilation. Therapy with digoxin maintains LV systolic function but does not prevent progressive LV enlargement.

Reversal of Chronic Molecular and Cellular Abnormalities Due to Heart Failure by Passive Mechanical Ventricular Containment

Abstract— Passive mechanical containment of failing left ventricle (LV) with the Acorn Cardiac Support Device (CSD) was shown to prevent progressive LV dilation in dogs with heart failure (HF) and increase ejection fraction. To examine possible mechanisms for improved LV function with the CSD, we examined the effect of CSD therapy on the expression of cardiac stretch response proteins, myocyte hypertrophy, sarcoplasmic reticulum Ca2+-ATPase activity and uptake, and mRNA gene expression for myosin heavy chain (MHC) isoforms. HF was produced in 12 dogs by intracoronary microembolization. Six dogs were implanted with the CSD and 6 served as concurrent controls. LV tissue from 6 normal dogs was used for comparison. Compared with normal dogs, untreated HF dogs showed reduced cardiomyocyte contraction and relaxation, upregulation of stretch response proteins (p21ras, c-fos, and p38 &agr;/&bgr; mitogen-activated protein kinase), increased myocyte hypertrophy, reduced SERCA2a activity with unchanged affinity for calcium, reduced proportion of mRNA gene expression for &agr;-MHC, and increased proportion of &bgr;-MHC. Therapy with the CSD was associated with improved cardiomyocyte contraction and relaxation, downregulation of stretch response proteins, attenuation of cardiomyocyte hypertrophy, increased affinity of the pump for calcium, and restoration of &agr;- and &bgr;-MHC isoforms ratio. The results suggest that preventing LV dilation and stretch with the CSD promotes downregulation of stretch response proteins, attenuates myocyte hypertrophy and improves SR calcium cycling. These data offer possible mechanisms for improvement of LV function after CSD therapy.

Chronic therapy with metoprolol attenuates cardiomyocyte apoptosis in dogs with heart failure.

OBJECTIVESnThe purpose of this study was to determine if therapy with beta-blockade is associated with reduced cardiomyocyte apoptosis.nnnBACKGROUNDnChronic treatment with beta-adrenergic blocking agents has been shown to improve left ventricular (LV) ejection fraction and attenuate progressive LV remodeling in heart failure (HF). Cardiomyocyte apoptosis has also been shown to occur in the failing heart.nnnMETHODSnModerate HF was produced in 14 dogs by intracoronary microembolizations. Dogs were randomized to three months therapy with metoprolol (MET, 25 mg twice daily, n = 7) or to no therapy at all (n = 7). At the end of three months, dogs were sacrificed, and nuclear DNA fragmentation (nDNAf), a marker of apoptosis, was assessed in LV tissue using the TUNEL assay. The number of cardiomyocytes with positive nDNAf labeling per 1,000 was quantified in LV regions bordering old infarcts and in regions remote from infarcts. Endonuclease activity and expression of the antiapoptotic protein Bcl-2 and the proapoptotic proteins Bax and caspase-3 were also evaluated in LV tissue.nnnRESULTSnThe number of nDNAf events per 1,000 cardiomyocytes was lower in dogs treated with MET compared with untreated dogs with HF in the border regions (0.35 +/- 0.07 vs. 5.32 +/- 0.77, p < 0.001) as well as the remote regions (0.07 +/- 0.05 vs. 0.39 +/- 0.12, p < 0.05). Endonuclease activity was also significantly lower in MET-treated compared with untreated dogs (25 +/- 3 vs. 37 +/- 2 ng [3H]DNA rendered soluble/min/mg protein). Western blotting for Bcl-2, Bax and caspase-3 showed increased expression of Bcl-2, decreased expression of caspase-3 and no change in Bax in MET-treated compared with untreated dogs.nnnCONCLUSIONSnChronic therapy with MET attenuates cardiomyocyte apoptosis in dogs with moderate HF. Attenuation of ongoing cardiomyocyte loss through apoptosis may be one mechanism through which beta-blockers elicit their benefits in HF.

Molecular mechanisms of reduced sarcoplasmic reticulum Ca2+ uptake in human failing left ventricular myocardium

BACKGROUNDnHuman failing heart due to idiopathic dilated cardiomyopathy is associated with decreased sarcoplasmic reticulum Ca(2+) uptake. However, it is unknown as to which mechanism leads to this abnormality.nnnMETHODSnImmunodetectable sarcoplasmic reticulum proteins (phospholamban [PLB], phosphorylated PLB at serine-16 or threonine-17, calsequestrin and Ca(2+)-ATPase levels), the activities of Ca(2+)-calmodulin-dependent protein kinase and protein phosphatase and Ca(2+) uptake at varying Ca(2+) concentrations were determined in left ventricular specimens from the same 7 failing hearts (ejection fraction 20 +/- 2%) due to idiopathic dilated cardiomyopathy and 5 non-failing explanted control donor hearts.nnnRESULTSnIn failing hearts, compared with control donors, decreased maximal velocity and affinity of Ca(2+) uptake for Ca(2+) were found to be associated with reduced expression levels of Ca(2+)-adenosine triphosphatase (ATPase), PLB and phosphorylated PLB at serine-16, but not of calsequestrin and phosphorylated PLB at threonine-17. In contrast, protein phosphatase activity increased significantly and the activity and protein expression level of the delta isoform of Ca(2+)-calmodulin-dependent protein kinase remained unchanged in failing hearts compared with control donors.nnnCONCLUSIONSnThe impaired maximal velocity of sarcoplasmic reticulum Ca(2+) uptake may be due in part to reduced protein expression level of Ca(2+)-ATPase, whereas the reduced affinity may be due in part to the reduced ratio of Ca(2+)-ATPase to PLB and reduced PLB phosphorylation at serine-16 in failing hearts. The latter abnormality may be due in part to increased protein phosphatase activity. These results suggest that selective enhancement of Ca(2+) uptake into the sarcoplasmic reticulum by pharmaceutical agents, or by molecular tools that inhibit phosphatase activity, would be a valuable therapeutic approach for treating, or at least retarding, the process of heart failure.

Interaction of β-adrenoceptor and adenosine receptor agonists on phosphorylation. Identification of target proteins in mammalian ventricles

The influence of the adenosine derivative (--)-N6-phenylisopropyladenosine (R-PIA, 1 micron) and 5N-ethylcarbox-amidoadenosine (NECA, 1 micron) on beta-adrenergic stimulated (isoproterenol, 10 nM) phosphorylation of sarcolemmal (15 kDa protein), sarcoplasmic reticular (phospholamban) and myofibrillar proteins (troponin I, C-protein) was studied in isolated 32P-labeled guinea-pig ventricles. The identification of the 15 kDa protein, phospholamban, troponin I and C-protein was based on their reaction with specific antibodies. Isoproterenol increased contractile parameters (developed tension, rate of tension development, rate of relaxation) and stimulated the phosphorylation state of a 15 kDa protein (now named phospholemman), of phospholamban, troponin I and C-protein (regarded as regulatory proteins). Isoproterenol concomitantly increased myocardial cyclic AMP levels. R-PIA and NECA attenuated the effects of isoproterenol on contractile parameters as well as on the phosphorylation of the regulatory proteins without affecting cyclic AMP levels. The effects of 1 microM R-PIA and 1 microM NECA on the isoproterenol-stimulated phosphorylation of regulatory proteins were blocked by the adenosine receptor antagonist 1.3-dipropyl-8-cyclopentylxanthine (DPCPX, 1 microM). Therefore, it is concluded that adenosine derivatives acting via adenosine receptors can reduce the isoproterenol-stimulated phosphorylation state of the following regulatory proteins: phospholemman, phospholamban, troponin I and C-protein.

Cardiac Contractility Modulation Electrical Signals Normalize Activity, Expression, and Phosphorylation of the Na+-Ca2+ Exchanger in Heart Failure

BACKGROUNDnExpression and phosphorylation of the cardiac Na(+)-Ca(2+) exchanger-1 (NCX-1) are up-regulated in heart failure (HF). We examined the effects of chronic cardiac contractility modulation (CCM) therapy on the expression and phosphorylation of NCX-1 and its regulators GATA-4 and FOG-2 in HF dogs.nnnMETHODS AND RESULTSnStudies were performed in LV tissue from 7 CCM-treated HF dogs, 7 untreated HF dogs, and 6 normal (NL) dogs. mRNA expression of NCX-1, GATA-4, and FOG-2 was measured using reverse transcriptase polymerase chain reaction, and protein level was determined by Western blotting. Phosphorylated NCX-1 (P-NCX) was determined using a phosphoprotein enrichment kit. Compared with NL dogs, NCX-1 mRNA and protein expression and GATA-4 mRNA and protein expression increased in untreated HF dogs, whereas FOG-2 expression decreased. Compared with NL dogs, the level of P-NCX-1 normalized to total NCX-1 increased in untreated HF dogs (0.80+/-0.10 vs 0.37+/-0.04; P < .05). CCM therapy normalized NCX-1 expression, GATA-4, and FOG-2 expression, and the ratio of P-NCX-1 to total NCX-1 (0.62+/-0.10).nnnCONCLUSIONnChronic monotherapy with CCM restores expression and phosphorylation of NCX-1. These findings are consistent with previous observations of improved LV function and normalized sarcoplasmic reticulum calcium cycling in the left ventricles of HF dogs treated with CCM therapy.

Effects of the AT1-receptor antagonist eprosartan on the progression of left ventricular dysfunction in dogs with heart failure.

We examined the effects of eprosartan, an AT1 receptor antagonist, on the progression of left ventricular (LV) dysfunction and remodelling in dogs with heart failure (HF) produced by intracoronary microembolizations (LV ejection fraction, EF 30 to 40%). Dogs were randomized to 3 months of oral therapy with low‐dose eprosartan (600 mg once daily, n=8), high‐dose eprosartan (1200 mg once daily, n=8), or placebo (n=8). In the placebo group, LV end‐diastolic (EDV) and end‐systolic (ESV) volumes increased after 3 months (68±7 vs 82±9 ml, P<0.004, 43±1 vs 58±7 ml, P<0.003, respectively), and EF decreased (37±1 vs 29±1%, P<0.001). In dogs treated with low‐dose eprosartan, EF, EDV, and ESV remained unchanged over the course of therapy, whereas in dogs treated with high‐dose eprosartan, EF increased (38±1 vs 42±1%, P<0.004) and ESV decreased (41±1 vs 37±1 ml, P<0.006), Eprosartan also decreased interstitial fibrosis and cardiomyocyte hypertrophy. We conclude that eprosartan prevents progressive LV dysfunction and attenuates progressive LV remodelling in dogs with moderate HF and may be useful in treating patients with chronic HF.

Muscarinic-cholinoceptor mediated attenuation of phospholamban phosphorylation induced by inhibition of phosphodiesterase in ventricular cardiomyocytes: Evidence against a cAMP- dependent effect

In intact guinea pig ventricles, acetylcholine (ACH) has been shown to attenuate the positive inotropic effects of isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, by reducing protein phosphorylation without altering cAMP levels. In the present study, we tested the hypothesis that the cAMP-independent inhibitory action of ACH is also evident in isolated cardiomyocytes. cAMP-dependent protein kinase (PKA) activity ratio (-cAMP/+cAMP) and phosphorylation of phospholamban (PLB) were determined in unlabeled and 32P-labeled guinea pig ventricular cardiomyocytes, respectively. IBMX increased PKA activity ratio and phosphorylation of PLB in a dose-dependent manner. When cardiomyocytes were incubated simultaneously with IBMX (0-1 mM) and ACH (2 μM), ACH attenuated PLB phosphorylation stimulated by low concentration (10-100 μM) but not by high concentrations (> 200 μM) of IBMX. EC50 value for IBMX-induced phosphorylation of PLB was 32 ± 6 μM and increased nearly 3-fold after addition of ACH while PKA activity ratio remained unchanged. The rank order of cyclic nucleotide derivatives to phosphorylate PLB was 8 bromo-cAMP > dibutyryl cAMP > 8 bromo-cGMP > dibutyryl cGMP. ACH reduced phosphorylation of PLB stimulated by 8 bromo-cAMP. We conclude that in isolated cardiomyocytes (1) ACH inhibits phosphorylation of PLB stimulated by either IBMX or 8 bromo-cAMP and (2) ACH does not lower IBMX-stimulated PKA activity ratio. These effects of ACH on PLB phosphorylation cannot be explained by a reduction in IBMX-stimulated cAMP levels but may involve the activation of protein phosphatases.

965-49 Abnormal Mitochondrial Respiration in Myocardium of Dogs with Chronic Heart Failure

We previously showed that abnormalities of mitochondria (M IT) exist in the failing heart and include hyperplasia, reduced organelle size and structural injury. In the present study, we examined MIT respiration in LV tissue obtained from 11 normal (NL) dogs and 8 dogs with heart failure (HF) produced by intracoronary microembolizations (LV ejection fraction 23xa0±xa03%). Tissue specimen (30xa0mg) were obtained from the subendocardial (ENDO) and subepicardial (EPI) halves of the LV wall. Basal (V o ) and state 3 (maximal) respiration (V ADP , after addition of 1xa0mM ADP) were measured with an oxygraph and Clark electrode using saponin skinned fiber bundles (0.2–0.3xa0mm). Respiratory rate was calculated in ngatoms of oxygen/min/mg of noncollagen protein. The respiration control ratio (RCR) was calculated as V ADP /V o . V o V ADP V ADP /V o ENDO EPI ENDO EPI ENDO EPI NL 9xa0±xa02 7xa0±xa01 46xa0±xa06 47xa0±xa01 6xa0±xa01 7xa0±xa01 HF 6xa0±xa01 6xa0±xa01 20xa0±xa05 22xa0±xa05 4xa0±xa01 4xa0±xa01 P-value l0.07 l0.5 l0.001 l0.005 l0.04 l0.004 MIT state 3 respiration is significantly reduced in myocardium of dogs with chronic HF. The observed reduction in the RCR confirms the presence of injury to inner MIT membrane. The abnormalities in MIT oxygen utilization support the concept of low energy production in the failing heart.