Hanumanth K. Reddy

Matrix metalloproteinase activity expression in infarcted, noninfarcted and dilated cardiomyopathic human hearts.

In the normal myocardium matrix metalloproteinases (MMP) are present in the latent form. To examine whether MMP are activated following infarction or idiopathic dilated cardiomyopathy (DCM), we extracted and measured MMP activity in tissue derived from 7 explanted, failing human hearts due to either previous myocardial infarction (MI) or DCM. MMP activity in infarcted left ventricle (LV), noninfarcted IV and right ventricle (RV) from MI patients, as well as tissue from either ventricle of DCM patients, were compared to the activity of donor heart tissue. SDS-PAGE and dye-binding assays were used to determine total protein concentration, while collagenase activity was measured by SDS-PAGE type substrate gels embedded with type I gelatin (zymography). Accuracy of the zymographic technique was shown for tissue samples as small as 0.05 mg and was comparable to results obtained by a spectrophotometric method.. After normalization for total protein concentration, we found 3 ± 1 % collagenase activity in normal atrial tissue which could be activated to 80–90% by trypsin or plasmin, indicating that collagenase is normally inactive or in a latent form in human heart. In endo- and epimyocardium of infarcted LV on the other hand, collagenase activity was 85–95% and 10–20%, respectively, while 5–10% and 3–5%, respectively, in noninfarcted LV In DCM, collagenolytic activity in the endo and epimyocardium was 75 ± 5 and 35 ± 5% in the LV and 35 ± 7 and 20 ± 5% in the RV, respectively. Thus, in dilated failing human hearts secondary to previous MI or DCM, MMP activity is increased. This is particularly the case within the endomyocardium of the infarcted and noninfarcted portions of either ventricle with MI and in both ventricles in DCM. This suggests that an activation of collagenase throughout the myocardium may contribute to its remodeling that includes ventricular dilatation and wall thinning.

Differential gene expression of extracellular matrix components in dilated cardiomyopathy

Extracellular matrix metalloproteinases (MMPs) are activated in dilated cardiomyopathic (DCM) hearts [Tyagi et al. (1996): Mol Cell Biochem 155:13‐21]. To examine whether the MMP activation is occurring at the gene expression level, we performed differential display mRNA analysis on tissue from six dilated cardiomyopathy (DCM) explanted and five normal human hearts. Specifically, we identified three genes to be induced and several other genes to be repressed following DCM. Southern blot analysis of isolated cDNA using a collagenase cDNA probe indicated that one of the genes induced during DCM was interstitial collagenase (MMP‐1). Northern blot analysis using MMP‐1 cDNA probe indicated that MMP‐1 was induced three‐ to fourfold in the DCM heart as compared to normal tissue. To analyze posttranslational expression of MMP and tissue inhibitor of matrix metalloproteinase (TIMP) we performed immunoblot, immunoassay, and substrate zymographic assays. TIMP‐1 and MMP‐1 levels were 37 ± 8 ng/mg and 9 ± 2 ng/mg in normal tissue specimens (P < 0.01) and 2 ± 1 ng/mg and 45 ± 11 ng/mg in DCM tissue (P < 0.01), respectively. Zymographic analysis demonstrated lytic bands at 66 kDa and 54 kDa in DCM tissue as compared to one band at 66 kDa in normal tissue. Incubation of zymographic gel with metal chelator (phenanthroline) abolished both bands suggesting activation of neutral MMP in DCM heart tissue. TIMP‐1 was repressed approximately twentyfold in DCM hearts when compared with normal heart tissue. In situ immunolabeling of MMP‐1 indicated phenotypic differences in the fibroblast cells isolated from the DCM heart as compared to normal heart. These results suggest disruption in the balance of myopathic‐fibroblast cell ECM‐proteinase and antiproteinase in ECM remodeling which is followed by dilated cardiomyopathy.

Myocardial fibrosis: role of ventricular systolic pressure, arterial hypertension, and circulating hormones

The myocardium contains myocyte and non-myocyte cells. A disproportionate growth of the nonmyocyte cell population can alter myocardial structure and lead to pathologic hypertrophy. Myocardial fibrosis, the result of cardiac fibroblast growth or abnormal accumulation of fibrillar collagen within the interstitial space, can adversely influence myocardial stiffness and ultimately ventricular function. We have examined the relative importance of ventricular systolic and arterial pressures and the effector hormones of the renin-angiotensin--aldosterone system in mediating this reactive fibrous tissue response in the hypertensive left and normotensive right ventricles in various experimental models of arterial hypertension. To date, our findings implicate arterial hypertension, together with an elevation in plasma aldosterone, as being contributory to the fibrosis in renovascular hypertension that creates tissue heterogeneity in either ventricle and impaired diastolic function. The endocrine properties of aldosterone in this nonclassical mineralocorticoid target tissue, the myocardium, requires further investigation.

Extracellular matrix regulation of metalloproteinase and antiproteinase in human heart fibroblast cells.

Following myocardial infarction, extracellular matrix (ECM) is disrupted, which leads to the generation of collagen‐ and elastin‐derived peptides (CDPs and EDPs, respectively). To investigate whether ECM‐derived peptides (i.e., CDPs and EDPs) induce extracellular proteinases in human heart fibroblast (HHF) cells, we isolated CDP and EDP using gelfiltration and antibody affinity column chromatography. The CDP and EDP were characterized by their intrinsic fluorescence due to cross‐link structure (pyridinoline and desmosine, respectively) and by immunoblot analysis using anti‐desmosine antibody. Neutrophil elastase and cathepsin G were identified using selective chromogenic substrates and by their specific inhibition with α1‐proteinase inhibitor and α1‐antichymotrypsin, respectively. Elastase and cathepsin G were elevated in the infarcted tissue. Selective inhibition of matrix metalloproteinase (MMP) by a higher concentration of tetracycline or doxycycline in zymographic gels elicited an inhibition constant (IC50) of 278 ± 10 μM and indicated that majority of MMP in the infarcted tissue is from fibroblast cells. The HHF proliferation was measured using an acid‐phosphatase assay. The EDP and CDP induce HHF cell proliferation. After EDP treatment phenotypic (formation of pseudopodia) changes were observed in HHF cells. To measure whether phenotypic changes by EDP or CDP are associated with MMP and tissue inhibitor of metalloproteinase (TIMP) expression in HHF cells, we measured MMP and TIMP expression by zymographic and Northern blot (mRNA) analyses. The expression of MMP and TIMP were upregulated at both the protein and gene transcription levels. These results suggested that during ischemic cardiomyopathy, initially neutrophil proteinase activates latent myocardial MMP which can degrade ECM, which continuously degrades if not controlled by TIMP, leading to ventricular dilatation and dysfunction.

Aspirin and clopidogrel hyporesponsiveness and nonresponsiveness in patients with coronary artery stenting.

Patients undergoing coronary artery stenting receive an antiplatelet regimen to reduce the risk of antithrombotic complications. Current guidelines recommend the use of acetyl salicylic acid (aspirin) and clopidogrel as evidenced by large clinical trials. There has been a concern about variable responses of patients to aspirin and clopidogrel which may predispose them to subacute stent thrombosis or late stent thrombosis. Up to 25% of patients with acute myocardial infarction (AMI) undergoing percutaneous coronary intervention (PCI) were found to have hyporesponsiveness or resistance to clopidogrel which may predispose them to recurrent events. Dual antiplatelet regimen is a standard therapy in these patients and there is always a concern about variable responses to aspirin and clopidogrel predisposing them to acute coronary syndrome (ACS). Prevalence of this hyporesponsiveness or resistance may be due to noncompliance, genetic mutations, co-morbid situations and concomitant use of other drugs. This issue is of considerable importance in the era of coronary drug eluting stents when a long-term dual antiplatelet regimen is needed. This paper is a review for clinicians taking care of such patients with hyporesponsiveness or nonresponsiveness to dual antiplatelet regimen.

Post-transplantation Lymphoproliferative Disorder in Heart and Kidney Transplant Patients: A Single-Center Experience

Background: Post-transplantation lymphoproliferative disorder (PTLD) after heart transplantation is a fatal complication, and standard treatment is either ineffective or too toxic. We have studied the incidence, clinical course, prognostic factors, and different treatment regimens pertaining to PTLD in 110 heart and 80 kidney transplant recipients. Methods: Information was abstracted from chart review of 110 heart transplant recipients and 80 kidney transplant recipients between January 1989 and October 2002. We report 15 patients with PTLD, 6 patients received a heart transplant and 9 patients received a renal transplant. Results: The overall incidence of PTLD was 8.9% (5.4% in heart and 13.7% in kidney transplant recipients). The average interval between transplantation and the diagnosis of PTLD in heart transplantation patients was 5.5 years, and their overall mean age was 44 years. The indications for transplantation were ischemic cardiomyopathy in 5 patients (1 patient received both heart and kidney transplants), glomerulonephritis in 6 patients, diabetes nephropathy in 2 patients, and polycystic disease in 2 patients. Six patients were diagnosed with early disease (<12 months), 7 with late onset (1 to 10 years), and 2 with very late onset (>10 years). Five patients had PTLD grade 2 (2 heart and 3 kidney transplants) and 10 patients had PTLD grade 3 (4 heart and 6 kidney transplants). Immunosuppressive treatment for PTLD patients consisted of cyclosporine, 73% (11/15); tacrolimus, 6.6% (1/15); prednisone, 100% (15/15); azathioprine, 80% (12/15); mycophenolate mofetil, 20% (3/15); murine monoclonal anti-human CD3 (OKT3), 7% (1/15); and anti-thymocyte globulin, 13% (2/15). PTLD developed in 11.5% of patients with primary Epstein-Barr virus infection and in 28.9% of patients with primary cytomegalovirus infection. Five patients received rituximab therapy, 5 had conventional chemotherapy, 3 had radiotherapy, 3 had reduction in immunosuppression, 2 had ganciclovir, 1 underwent surgery, and 1 patient died before receiving treatment. The mortality rate was 26.6%. The average interval between transplantation and the diagnosis of PTLD in heart transplant recipients was 5.5 years. The mortality rate was significantly higher in the control group than in the rituximab group. Conclusions: Caucasian race and male gender were independent risk factors for developing PTLD. Pretransplant cytomegalovirus seropositive status is a strong predictor of developing PTLD. Management of PTLD requires randomized controlled trials of various chemotherapeutic and antiviral drugs regimens. Treatment of PTLD with rituximab is a beneficial alternative with a favorable outcome. Patients in whom primary Epstein-Barr virus, cytomegalovirus, or hepatitis C infection develop after transplantation should be managed with heightened surveillance for the development of PTLD. Further randomized trials are needed to evaluate the efficacy of antiviral drugs, intravenous immunoglobulin, interferon, and prophylactic Epstein-Barr virus immunization strategies.

Thinking beyond low-density lipoprotein cholesterol: strategies to further reduce cardiovascular risk

Several large statin trials and meta-analyses have demonstrated a reduction in low-density lipoprotein cholesterol (LDL-C) and cardiovascular morbidity and mortality. Some trials have also highlighted the significance of residual cardiovascular risk after treatment of LDL-C to target levels. This reflects the complex nature of residual cardiovascular risk. This residual risk is partially due to low HDL-C and high triglycerides (TG) despite achievement of LDL goals with statin therapy. The NCEP ATP III guidelines reported that low HDL-C is a significant and an independent risk factor for coronary heart disease (CHD) and is inversely related to CHD. Epidemiologic studies have also shown a similar inverse relationship of HDL-C with CHD. High-density lipoprotein cholesterol (HDL-C) may directly participate in the anti-atherogenic process by promoting efflux of cholesterol of the foam cells of atherogenic lesions. Many studies have demonstrated multiple anti-atherogenic actions of HDL-C and its role in promoting efflux of cholesterol from the foam cells. The residual risk by increased TG with or without low HDL-C can be assessed by calculating non–HDL-C and a reduction in TG results in decreased CHD.

Collagen cross-linking: New dimension to cardiac remodeling

See article by Badenhorst et al. [1] (pages 632–641) in this issue. Left ventricular (LV) dysfunction has become a leading area of research as the prevalence of heart failure is reaching epidemic proportions. LV chamber remodeling and stiffness are consequent to significant structural and functional alteration of both the myocyte and extracellular matrix (ECM). Both these factors are important determinants of chamber size and geometry as well as its contractile and relaxation properties. Numerous papers have been published about the seminal role of collagen concentration and collagenolysis in hypertrophic and dilated cardiomyopathies; however, the qualitative aspects of these changes were not correlated well with the functional characteristics of cardiac chambers. In this issue of Cardiovascular Research , Baldenhorst et al. [1] show the influence of collagen cross-linking on chamber stiffness and remodeling. Cellular mechanisms responsible for transformation of compensatory myocardial hypertrophy to a dysfunctional dilated ventricle remain enigmatic. Earlier studies focused on structural changes in the cardiac myocyte to explain chamber dysfunction in hypertensive heart disease [2–5]. This included studies related to changes in its size and spatial orientation [2–4]. The depression of systolic function and an increase in passive stiffness were noted even in isolated muscle preparations [5]. This suggested that physiological changes in the myocardial chamber were related to structural changes at the cardiac myocyte level. More recent studies have focused on the contribution of the ECM to cardiac contraction and relaxation functions [6–11]. The collagen matrix provides support for the maintenance of both myocyte and myofibrillar alignment, thereby ensuring structural integrity for individual myocyte shortening and relaxation, which then translates into overall myocardial systolic and diastolic properties. Therefore, the patterns of collagen synthesis and degradation play a seminal role in determining the altered systolic and diastolic properties of a remodeled ventricle in response … * Corresponding author. DC 034.00, Division of Cardiology, University of Missouri—Columbia, 1 Hospital Drive, Columbia, MO 65212, USA. Tel.: +1-573-882-2296; fax: +1-573-884-7743.

Increased Endothelin-Induced Ca2+ Signaling, Tyrosine Phosphorylation, and Coronary Artery Disease in Diabetic Dyslipidemic Swine Are Prevented by Atorvastatin

Endothelin-1 (ET-1) signaling mechanisms have been implicated in the pathogenesis of excess coronary artery disease in diabetic dyslipidemia. We hypothesized that in diabetic dyslipidemia ET-1-induced coronary smooth muscle calcium (Ca2+m) and tyrosine phosphorylation would be increased, and the lipid lowering agent, atorvastatin, would inhibit these increases. Male Yucatan miniature swine groups were treated for 20 weeks: normal low-fat fed control, high-fat/cholesterol fed (hyperlipidemic), hyperlipidemic made diabetic with alloxan (diabetic dyslipidemic), and diabetic dyslipidemic treated with atorvastatin (atorvastatin-treated). Blood glucose values were 5-fold greater in diabetic dyslipidemic and atorvastatin-treated versus control and hyperlipidemic. Total and low-density lipoprotein (LDL) plasma cholesterol in hyperlipidemic, diabetic dyslipidemic, and atorvastatin-treated were ∼5-fold greater than control. Intravascular ultrasound detectable coronary disease and hypertriglyceridemia were only observed in diabetic dyslipidemic and were abolished by atorvastatin. In freshly isolated cells, the Ca2+m response to ET-1 in diabetic dyslipidemic was greater than in control, hyperlipidemic, and atorvastatin-treated groups. Selective ET-1 receptor antagonists showed in the control group that the ETB subtype inhibits ETA regulation of Ca2+m. There was almost a complete switch of receptor subtype regulation of Ca2+m from largely ETA in control to an increased inhibitory interaction between ETA and ETB in hyperlipidemic and diabetic dyslipidemic groups, such that neither ETA nor ETB antagonist alone could block the ET-1-induced Ca2+m response. The inhibitory interaction was attenuated in the atorvastatin-treated group. In single cells, basal and ET-1-induced tyrosine phosphorylation in diabetic dyslipidemic were more than 3- and 6-fold greater, respectively, than in control, hyperlipidemic, and atorvastatin-treated. Attenuation by atorvastatin of coronary disease and ET-1-induced Ca2+m and tyrosine phosphorylation signaling with no change in cholesterol provides strong evidence for direct actions of atorvastatin and/or triglycerides on the vascular wall.

Myocardial energetics and efficiency in patients with idiopathic cardiomyopathy: response to dobutamine and amrinone.

Nine consecutive patients having severe idiopathic dilated cardiomyopathy were studied for their response in ventricular function, coronary sinus blood flow and myocardial oxygen consumption, lactate extraction and efficiency following incremental doses of dobutamine, followed by the combination of dobutamine and the phosphodiesterase inhibitor amrinone. Results, presented as baseline and the response to the peak dose (15 micrograms/kg/min) of dobutamine and to the combination of dobutamine and amrinone (each at 15 micrograms/kg/min) (differences compared with baseline) were: wedge pressure decreased from 28 +/- 7 to 26 +/- 8 mm Hg (p = NS) and to 20 +/- 6 mm Hg (p less than 0.01); cardiac index rose from 1.47 +/- 0.44 L/min/m2 to 2.89 +/- 1.1 L/min/m2 (p less than 0.01) and to 3.64 +/- 1.05 L/min/m2 (p less than 0.001); myocardial oxygen consumption remained invariant (18 +/- 8, 17 +/- 5, and 19 +/- 5 ml/min) despite progressive increments in minute work from 2.96 +/- 1.1 to 6.98 +/- 3.9 kg - m/min (p less than 0.01) and to 9.38 +/- 4.3 kg - m/min (p less than 0.001); myocardial lactate extraction rose from 21 +/- 10% to 30 +/- 15% (p = NS) and to 35 +/- 10% with the addition of amrinone (p less than 0.01). No patient had net lactate efflux into the coronary sinus, and myocardial efficiency improved from 9.5 +/- 5% to 21.7 +/- 13.0% (p less than 0.01) and to 28.0 +/- 18.0% (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)