Victor G. Sharov

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.

Progression of heart failure: A role for interstitial fibrosis

Progressive deterioration of left ventricular (LV) function is a characteristic feature of the heart failure (HF) state. The mechanism or mechanisms responsible for this hemodynamic deterioration are not known but may be related to progressive intrinsic dysfunction, degeneration and loss of viable cardiocytes. In the present study, we tested the hypothesis that accumulation of collagen in the cardiac interstitium (reactive interstitial fibrosis, RIF), known to occur in HF, results in reduced capillary density (CD=capillary/fiber ratio) and increased oxygen diffusion distance (ODD) which can lead to hypoxia and dysfunction of the collagen encircled myocyte. Studies were performed in LV tissue obtained from 10 dogs with chronic HF (LV ejection fraction 26±1%) produced by multiple sequential intracoronary, microembolizations. In each dog, CD and ODD were evaluated in LV regions that manifested severe RIF (volume fraction 16±2%) and in LV regions of little or no RIF (volume fraction 4±1%). In regions of severe RIF, CD was significantly decreased compared to regions of no RIF (0.92±0.02 vs. 1.05±0.03) (P<0.03). Similarly, ODD was significantly increased in regions of severe RIF compared to regions of no RIF (15.3±0.4 vs. 12.2±0.3 μm) (P<0.001). These data suggest that in dogs with chronic HF, constituent myocytes of LV regions which manifest severe RIF may be subjected to chronic hypoxia; a condition that can adversely impact the function and viability of the collagen encircled cardiocyte.

Mitochondrial abnormalities in myocardium of dogs with chronic heart failure

The number, size and structural integrity of mitochondria (MIT) were evaluated in the myocardium of 12 dogs with chronic heart failure (CHF) produced by sequential intracoronary microembolizations (EMB). Tissue specimens for transmission electron microscopy were obtained from the left ventricular (LV) free wall, septum and right ventricular free wall 3 to 4 months after the last EMB. Comparisons were made with samples obtained from identical sites in 9 control dogs. In dogs with CHF, LV ejection fraction decreased from 61 +/- 1% at baseline (prior to EMB) to 22 +/- 2% 3 to 4 months after the last EMB (P < 0.01) while plasma norepinephrine (PNE) concentration increased from 364 +/- 12 pg/ml to 837 +/- 150 pg/ml (P < 0.01). The number of MIT in an area of 100 square sarcomeres was greater in CHF dogs compared to controls (92 +/- 5 vs 64 +/- 2) (P < 0.001); whereas the average size of MIT was smaller (0.53 +/- 0.03 vs. 0.78 +/- 0.04 microm2) (P < 0.001). Injury ranging in severity from matrix depletion to myelinization and membrane disruption was present in 27 +/- 4% of MIT of CHF dogs compared to only 3 +/- 1% of MIT of controls (P < 0.001). MIT abnormalities were present to the same extent in all three regions of the heart. The severity of MIT injury, assessed on the basis of an injury index, was significantly higher in CHF dogs with PNE > or = 600 pg/ml (0.64 +/- 0.07) compared to CHF dogs with PNE < 600 pg/ml (0.32 +/- 0.08) (P < 0.01). Among CHF dogs, the MIT injury index was linearly related to PNE concentration (r = 0.57, P < 0.05), LV ejection fraction (r = 0.57, P < 0.05) and LV end-diastolic pressure (r = 0.57, P < 0.05). These data indicate that profound MIT abnormalities are present in the myocardium of dogs with CHF and are related to PNE concentration and to the severity of LV dysfunction.

Apoptosis in heart failure

A characteristic feature of heart failure is the progressive worsening of ventricular function over months or years despite the absence of clinically apparent intercurrent adverse events. The mechanism or mechanisms responsible for this hemodynamic deterioration are not known but may be related to progressive intrinsic contractile dysfunction of residual viable cardiac myocytes, or to ongoing degeneration and loss of myocytes, or both. This report will address the concept of ongoing cardiac myocyte loss that may occur during the course of evolving heart failure viewed from the perspective of apoptosis or programmed cell death as the potential mediator of cardiac muscle cell loss. In recent years, several studies have shown that constituent myocytes of failed explanted human hearts and hearts of animals with experimentally induced heart failure undergo apoptosis. Recent studies have shown that cardiac myocyte apoptosis also occurs after acute myocardial infarction, as well as in the hypertrophied heart and the aging heart, conditions frequently associated with the development of heart failure. Considerable work has also been conducted and novel concepts advanced to explain potential molecular triggers of cardiac myocyte apoptosis in heart failure. Although available data support the existence of myocyte apoptosis in the failing heart, questions essential to our understanding of the importance of myocyte apoptosis in this disease process remain unanswered. Lacking are studies aimed at identifying physiological factors inherent to heart failure that trigger myocyte apoptosis. Also lacking are studies that address the importance of myocyte apoptosis in the progression of left ventricular dysfunction. If loss of cardiac myocytes through apoptosis can be shown to be an important contributor to the progression of heart failure, and if factors that trigger apoptosis in the heart can be identified, such knowledge can potentially lead to the development of novel therapeutic modalities aimed at preventing, or at the very least retarding, the process of progressive ventricular dysfunction and the ultimate transition toward end-stage, intractable heart failure.

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.

Decreased proportion of type I myofibers in skeletal muscle of dogs with chronic heart failure.

BackgroundWhether biochemical and histological abnormalities of skeletal muscle (SM) develop in patients with chronic heart failure (HF) remains controversial. In the present study, dogs with chronic HF were used to examine potential alterations of SM fiber type, fiber size, number of capillaries per fiber (C/F), B-adrenergic receptor density (Bmax), and fiber ultrastructural integrity. Methods and ResultsHF was produced in 17 dogs by sequential intracoronary microembolizations. Biopsies of the lateral head of the triceps muscle were used in all studies. Type I and type II fibers were differentiated by myofibrillar ATPase (pH 9.4 or 4.2). Bmax was assessed by radioligand binding and SM ultrastructure by transmission electron microscopy. Comparisons were made with biopsies obtained from nine control dogs. The percentage of SM type I fibers was reduced in HF dogs compared with control dogs (19±2% versus 32±5%) (p<0.001), whereas the percentage of SM type II fibers was increased (81±2% versus 68±5%) (p<0.001). The change in fiber type composition was not associated with a preferential atrophy or hypertrophy of either fiber type. There was no difference in SM Bmax (198.9+14.3 versus 186.8±17.3 fmol/mg protein) or in C/F (537±0.26 versus 5.84±0.21) between HF dogs and control dogs. No ultrastructural abnormalities were present in SM fibers of HF dogs. ConclusionsIn dogs with HF, there is a decrease in the relative composition of the slow-twitch type I SM fibers and an increase in fast-twitch type II fibers. The shift in fiber type composition is not associated with preferential atrophy of either fiber type or with a reduction in C/F, B-adrenergic receptor density, or structural abnormalities of the myofibers.

Cell Death, Tissue Hypoxia and the Progression of Heart Failure

An important feature of heart failure is the progressive deterioration of left ventricular function that occurs in the absence of clinically apparent intercurrent adverse events. The mechanism or mechanisms responsible for this hemodynamic deterioration are not known. We and others have advanced the hypothesis that this hemodynamic deterioration results from progressive intrinsic contractile dysfunction of viable cardiomyocytes and/or from ongoing loss of cardiomyocytes. This review will focus on the concept of ongoing cardiac myocyte loss as a contributing factor to the progression of left ventricular dysfunction that characterizes the heart failure state. Specifically, the discussion will center on apoptosis or “programmed cell death” as a potential mediator of cardiomyocyte loss. In recent years, several studies have shown that constituent myocytes of failed explanted human hearts and hearts of animals with experimentally induced heart failure undergo apoptosis. Studies have also shown that cardiomyocyte apoptosis occurs following acute myocardial infarction, in the hypertrophied heart as well as in the aging heart; conditions frequently associated with the development of failure. While available data support the existence of myocyte apoptosis in the failing heart, lacking are studies which address the importance of myocyte apoptosis in the progression of LV dysfunction. As part of this discussion, we will address this issue and construct a case in support of a concept that the failing myocardium is subject to regional hypoxia, an abnormality that can potentially trigger cardiomyocyte apoptosis. If loss of cardiac myocytes through apoptosis can be shown to be an important contributor to the progression of heart failure, and if exact physiologic and molecular factors that trigger apoptosis in the heart can be identified, the stage will be set for the development of novel therapeutic modalities aimed at preventing, or at the very least retarding, the process of progressive ventricular dysfunction and the ultimate transition toward end-stage, intractable heart failure.

Effects of long-term therapy with bosentan on the progression of Left ventricular dysfunction and remodeling in dogs with heart failure

OBJECTIVESnIn this study, we examined the effects of long-term therapy with bosentan on the progression of LV dysfunction and remodeling in dogs with moderate HF.nnnBACKGROUNDnAcute intravenous administration of bosentan, a mixed endothelin-1 type A and type B receptor antagonist, was shown to improve left ventricular (LV) function in patients and dogs with heart failure (HF).nnnMETHODSnLeft ventricular dysfunction was induced by multiple, sequential intracoronary microembolizations in 14 dogs. Embolizations were discontinued when LV ejection fraction (EF) was between 30% and 40%. Dogs were randomized to three months of therapy with bosentan (30 mg/kg twice daily, n = 7) or no therapy at all (control, n = 7).nnnRESULTSnIn untreated dogs, EF decreased from 35 +/- 1% before initiating therapy to 29 +/- 1% at the end of three months of therapy (p = 0.001), and LV end-diastolic volume (EDV) and end-systolic volume (ESV) increased (EDV: 71 +/- 3 vs. 84 +/- 8 ml, p = 0.08; ESV: 46 +/- 2 vs. 60 +/- 6 ml, p = 0.03). By contrast, in dogs treated with bosentan, EF tended to increase from 34 +/- 2% before initiating therapy to 39 +/- 1% at the end of three months of therapy (p = 0.06), and EDV and ESV decreased (EDV: 75 +/- 3 vs. 71 +/- 4 ml, p = 0.05; ESV: 48 +/- 2 vs. 43 +/- 3 ml, p = 0.01). Furthermore, compared with untreated dogs, dogs treated with bosentan showed significantly less LV cardiomyocyte hypertrophy and LV volume fraction of interstitial fibrosis.nnnCONCLUSIONSnIn dogs with moderate HF, long-term therapy with bosentan prevents the progression of LV dysfunction and attenuates LV chamber remodeling. The findings support the use of mixed endothelin-1 receptor antagonists as adjuncts to the long-term treatment of HF.