Richard E. Sievers

Heat-shock protein induction in rat hearts. A direct correlation between the amount of heat-shock protein induced and the degree of myocardial protection.

BACKGROUND Previous studies have demonstrated that heat-shock treatment results in the induction of 72-kD heat-shock protein (HSP72) and a reduction of infarct size after subsequent ischemia and reperfusion. METHODS AND RESULTS To test the hypothesis that the degree of protection from ischemic injury in heat-shocked rats correlates with the degree of prior HSP72 induction, rats pretreated with 40 degrees C, 41 degrees C, or 42 degrees C of whole-body hyperthermia followed by 24 hours of recovery and control rats (n = 6 in each group) were quantitatively assessed for the presence of myocardial HPS72 by optical densitometry of Western blots and a primary antibody that is specific for HSP72 and a tertiary antibody labeled with 125I. Although rats heat-shocked to 40 degrees C had no significant induction of myocardial HSP72, rats heat-shocked to 41 degrees C and 42 degrees C demonstrated progressively increased amounts of myocardial HSP72 compared with controls. Separate groups of rats heat-shocked to 40 degrees C (n = 16), 41 degrees C (n = 37), and 42 degrees C (n = 36) with 24 hours of recovery and controls (n = 26) were subjected to 35 minutes of left coronary artery occlusion and 120 minutes of reperfusion. Compared with control and 40 degrees C rats, there was progressive infarct size reduction, assessed by triphenyltetrazolium chloride staining, in rats that were heat-shocked to 41 degrees C and 42 degrees C. Furthermore, there was a direct correlation between the amount of HSP72 induced and the reduction in infarct size (r = .97, P = .037). CONCLUSIONS These results suggest that the improved salvage after heat-shock pretreatment may be related to the amount of HSP72 induced before prolonged ischemia and reperfusion.

Fibrin Glue Alone and Skeletal Myoblasts in a Fibrin Scaffold Preserve Cardiac Function after Myocardial Infarction

Current efforts in cardiac tissue engineering center around the use of scaffolds that deliver cells to the epicardial surface. In this study, we examined the effects of fibrin glue as an injectable scaffold and wall support in ischemic myocardium. The left coronary artery of rats was occluded for 17 min, followed by reperfusion. Echocardiography was performed 8 days after infarction. One to 2 days later, either 0.5% bovine serum albumin (BSA) in phosphate-buffered saline, fibrin glue alone, skeletal myoblasts alone, or skeletal myoblasts in fibrin glue were injected into the ischemic left ventricle. Echocardiography was again performed 5 weeks after injection. The animals were then sacrificed and the hearts were fresh frozen and sectioned for histology and immunohistochemistry. Both the fractional shortening (FS) and infarct wall thickness of the BSA group decreased significantly after 5 weeks (p = 0.0005 and 0.02, respectively). In contrast, both measurements for the fibrin glue group, cells group, and cells in fibrin glue group did not change significantly (FS: p = 0.18, 0.89, and 0.19, respectively; wall thickness: p = 0.40, 0.44, 0.43, respectively). Fibrin glue is capable of preserving infarct wall thickness and cardiac function after a myocardial infarction in rats and may be useful as a biomaterial scaffold for myocardial cell transplantation.

Nuclear Magnetic Resonance Imaging of Acute Myocardial Infarction in Dogs: Alterations in Magnetic Relaxation Times

Nuclear magnetic resonance (NMR) imaging was used to study 24-hour-old acute myocardial infarctions in 8 dogs. Images and measurements of excised hearts were obtained in a 6.5 ml bore-resistive NMR imager (0.35 Tesla). Spin echo NMR imaging in each instance demonstrated the area of infarction as a region of increased signal intensity compared with that in normal myocardium. The T1 and T2 values of the area of infarction were greater than those of normal myocardium in all dogs. For each dog the T1 value was greater for the infarct region; however, the group mean value for T1 (ms) of the infarct region (728 +/- 94) was not significantly greater than that for the normal region (650 +/- 87). The T2 value (ms) was discriminate for all dogs, and the mean value for the infarct region (48 +/- 2) was significantly different (p less than 0.01) from the value for normal myocardium (42 +/- 1). The percent water content of the infarct (79 +/- 1%) was significantly greater (p less than 0.01) than that of normal regions (76 +/- 1%). The linear relationship between T2 value and percent water content showed a good correlation coefficient (r = 0.90; p less than 0.01). NMR imaging detects acute myocardial infarction as a positive image without contrast media. Increased signal intensity of the infarct is related to increased hydrogen density and increased T2 relaxation time.

Heat shock protein induction in rat hearts. A role for improved myocardial salvage after ischemia and reperfusion

BackgroundTo test the hypothesis that the heat shock response is associated with improved myocardial salvage after myocardial ischemia and reperfusion, rats treated with prior whole-body hyperthermia and 24 hours of recovery (n=26) or 20 minutes of ischemic pretreatment and 8 hours of recovery (n=24) and control rats (n=27, n=24, for hyperthermic and ischemic pretreatment, respectively) were subjected to 35 minutes of left coronary artery (LCA) occlusion and 120 minutes of reperfusion. Methods and ResultsAlthough ventricular samples from rats subjected to either hyperthermia (n=7) or ischemic pretreatment (n=6) all showed induction of HSP72 (heat shock protein), Western blot analysis revealed significantly greater amounts of HSP72 in samples obtained from rats subjected to hyperthermia compared with those from rats subjected to ischemic pretreatment. Control rats (n =7) showed no significant presence of myocardial HSP72. After 35 minutes of LCA occlusion and 2 hours of reperfusion, infarct size was significantly reduced in heat-shocked rats compared with controls (8.4±1.7%, n=26 versus 15.5±1.99, n=27; p=0.007; mean±SEM; infarct mass/left ventricular massx 100). There were no significant differences in left ventricular (LV) systolic pressure, heart rate, LV dP/dt, or rate-pressure product between heat-shocked (n=11) and control (n=14) rats during the ischemic period. There were no differences in infarct size between ischemically pretreated and control rats subjected to 35 minutes of ischemia and reperfusion (9.7±2.1%, n=23 versus 10.0±2.1, n=24; p=NS). ConclusionsIn this model of ischemia and reperfusion, prior heat shock was associated with significantly improved myocardial salvage after 35 minutes of LCA occlusion and reperfusion. This improved salvage was correlated with marked HSP72 induction and was independent of the hemodynamic determinants of myocardial oxygen supply and myocardial oxygen demand during the ischemic period. In contrast, mild HSP72 induction by ischemic pretreatment was not associated with improved myocardial salvage after myocardial ischemia and reperfusion. Thus, the absolute levels of HSP72 may be important in conferring protection from ischemic injury in this animal model.

Overexpression of Heat Shock Protein 72 in Transgenic Mice Decreases Infarct Size In Vivo

BACKGROUND Previous studies have demonstrated that induction of heat shock protein (HSP) 72 by whole-body hyperthermia reduces infarct size in an in vivo model of ischemia and reperfusion. Furthermore, hearts obtained from transgenic mice that overexpress HSP72 demonstrate improved functional recovery and decreased infarct size in vitro after global ischemia and reperfusion. METHODS AND RESULTS To test the hypothesis that overexpression of HSP72 in transgenic mice reduces infarct size in vivo, transgenic mice that were heterozygous for a rat HSP70i gene ([+]HSP72) and transgene-negative littermate controls ([-]HSP72) were subjected to 30 minutes of left coronary artery occlusion followed by 120 minutes of reperfusion. Core body temperature was monitored with a rectal thermometer and maintained between 36.5 degrees C and 37.0 degrees C with a heating pad. Infarct size, determined by dual staining with triphenyltetrazolium chloride and phthalocyanine blue dye, was smaller in [+]HSP72 mice compared with [-]HSP72 mice (12.7 +/- 2.8% [n = 7] versus 33.4 +/- 4.5% [n = 6], infarct size/risk area, respectively; P < .05; mean +/- SEM). CONCLUSIONS Overexpression of HSP72 reduces infarct size in this in vivo transgenic mouse model of myocardial ischemia and reperfusion.

Loss of myocardial protection after preconditioning correlates with the time course of glycogen recovery within the preconditioned segment.

BackgroundAlthough previous investigators have demonstrated that myocardial preconditioning reduces infarct size, the mechanisms of cardioprotection associated with preconditioning are not completely understood. Methods and ResultsTo test the hypothesis that preconditioning (four 5-minute episodes of ischemia each followed by 5 minutes of reperfusion) reduces infarct size by depleting cardiac glycogen stores and attenuating the degree of intracellular acidosis during subsequent prolonged left coronary artery occlusion, preconditioned and control rats were subjected to 45 minutes of left coronary artery occlusion and 120 minutes of reflow immediately after preconditioning (groups 1P and 1C, respectively) or after 30 minutes (groups 2P+30m and 2C), 1 hour (groups 3P+60m and 3C), or 6 hours (groups 4P+360m and 4C) of nonischemic recovery after preconditioning but before prolonged ischemia. In each group, cardiectomy was performed in selected rats immediately before prolonged ischemia for cardiac glycogen assay. In selected animals, 31P magnetic resonance spectroscopy was performed to monitor intracellular pH and measure high-energy phosphate levels during ischemia and reperfusion. Group 1P rats demonstrated marked glycogen depletion after preconditioning compared with controls (0.72±039 [n=91 versus 5.67±1.73 [n=12] mg glucose/g wet wt; p<0.001 versus group 1C) that was associated with attenuation of intracellular acidosis during ischemia, as measured by 31P magnetic resonance spectroscopy (6.8±03 [n=111 versus 6.2±03 [n=91 pH units; p<0.01), and marked infarct size reduction (03±0.6% [n=7] versus 38.1±113% [n=71, infarct size divided by risk area; p<0.0001). During ischemia, there were no differences in myocardial ATP or phosphocreatine levels or in any hemodynamic determinant of myocardial oxygen demand between groups 1P and 1C. In preconditioned rats that were allowed to recover before ischemia (groups 2P+30m, 3P+60m, and 4P+360m), the time course of glycogen repletion paralleled the loss of protection from ischemic injury. ConclusionsGlycogen depletion and the attenuation of intracellular acidosis during ischemia appear to be important factors in delaying irreversible injury and reducing infarct size in this animal model of myocardial preconditioning.

The use of human mesenchymal stem cells encapsulated in RGD modified alginate microspheres in the repair of myocardial infarction in the rat.

The combination of scaffold material and cell transplantation therapy has been extensively investigated in cardiac tissue engineering. However, many polymers are difficult to administer or lack the structural integrity to restore LV function. Additionally, polymers need to be biological friendly, favorably influence the microenvironment and increase stem cell retention and survival. This study determined whether human mesenchymal stem cells (hMSCs) encapsulated in RGD modified alginate microspheres are capable of facilitating myocardial repair. The in vitro study of hMSCs demonstrated that the RGD modified alginate can improve cell attachment, growth and increase angiogenic growth factor expression. Alginate microbeads and hMSCs encapsulated in microbeads successfully maintained LV shape and prevented negative LV remodeling after an MI. Cell survival was significantly increased in the encapsulated hMSC group compared with PBS control or cells alone. Microspheres, hMSCs, and hMSCs in microspheres groups reduced infarct area and enhanced arteriole formation. In summary, surface modification and microencapsulation techniques can be combined with cell transplantation leading to the maintenance of LV geometry, preservation of LV function, increase of angiogenesis and improvement of cell survival.

Ischemic Preconditioning Decreases Apoptosis in Rat Hearts In Vivo

BACKGROUND Previous studies have demonstrated that ischemic preconditioning prevents lethal cell injury and, as a consequence, limits infarct size in rat heart. Although both apoptosis and necrosis have been shown to contribute to myocardial cell death after myocardial ischemia and reperfusion, the ability of ischemic preconditioning to prevent programmed cell death remains unknown. METHODS AND RESULTS To test the hypothesis that ischemic preconditioning reduces irreversible ischemic injury in part by decreasing apoptosis, rats that underwent ischemic preconditioning and controls were subjected to 30 minutes of left coronary artery occlusion followed by 180 minutes of reperfusion. Ischemic preconditioning was achieved by five 5-minute cycles of ischemia, each followed by 5 minutes of reperfusion. Infarct size, determined by dual staining with triphenyltetrazolium chloride and phthalocyanine blue dye, was significantly reduced in preconditioned compared with nonpreconditioned rats (11.4+/-1.4% versus 58.7+/-1.4%; n=20 in each group; P<.001; infarct size/risk area). Genomic DNA from preconditioned hearts showed little or no oligonucleosome-sized fragments (200-bp multiples), whereas genomic DNA from nonpreconditioned hearts showed a typical nucleosome fragmentation. The TUNEL assay localized fewer and sparsely stained nuclei within the infarct zone of ischemic preconditioned hearts compared with nonpreconditioned hearts. Consistent with these findings, the number of cytosolic histone-associated low-molecular-weight DNA fragments was significantly decreased in preconditioned hearts compared with controls (0.17+/-0.02 versus 1.07+/-0.09 U; n=10 in each group; P<.001; absorbance 405 nm/490 nm). CONCLUSIONS This study suggests that ischemic preconditioning reduces irreversible ischemic injury in part by decreasing apoptosis after prolonged ischemia and reperfusion.

Verapamil Suppresses Atherosclerosis in Cholesterol-Fed Rabbits

The effect of verapamil, a drug that reduces the concentration of intracellular calcium, on atherogenesis was evaluated in rabbits fed a cholesterol-rich diet for 10 weeks. Ten rabbits received oral verapamil, 8 mg/kg daily; eight received the same oral dose and 0.5 mg/kg daily subcutaneously; nine received oral lanthanum, 35 mg/kg daily, and nine were controls. Over the 10 week period, all groups had average serum cholesterol levels greater than 1,500 mg/dl (normal = 90 +/- 63 mg/dl). At the end of the experiment, the aortas were removed, opened and stained for lipid with Sudan IV. The extent of atherosclerosis was determined by planimetry. The group receiving oral and parenteral verapamil had significantly less atherosclerosis (25 +/- 26% of total intimal area; mean +/- standard deviation), as compared with the controls (73 +/- 24%). Reduction of atherosclerosis with oral verapamil (51 +/- 22%) and lanthanum (59 +/- 31) was not statistically significant. Indexes of contractility in isolated right ventricular papillary muscles (developed tension at maximal length [Lmax] and maximal velocity of shortening [Vmax]) were reduced in the group treated with oral and parenteral verapamil, but not in the others. It is concluded that verapamil suppresses the development of atherosclerosis in rabbits fed a cholesterol-rich diet.

The effect of injected RGD modified alginate on angiogenesis and left ventricular function in a chronic rat infarct model.

Congestive heart failure (CHF) is a chronic disease with a high mortality rate. Managing CHF patients has been one of the most severe health care problems for years. Scaffold materials have been predominantly investigated in acute myocardial infarction (MI) studies and have shown promising improvement in LV function. In this study we examined whether surface modification of a biomaterial can influence the myocardial microenvironment and improve myocardial function in a rodent model of ischemic cardiomyopathy. In vitro cell culture and in vivo rat studies were performed. RGD peptides conjugated to alginate improved human umbilical vein endothelial cell (HUVEC) proliferation and adhesion when compared to a non-modified alginate group. Injection of the alginate hydrogel into the infarct area of rats 5 weeks post-MI demonstrated that both modified and non-modified alginate improve heart function, while LV function in the control group deteriorated. Both the RGD modified alginate and non-modified alginate increased the arteriole density compared to control, with the RGD modified alginate having the greatest angiogenic response. These results suggest that in situ use of modified polymers may influence the tissue microenvironment and serve as a potential therapeutic agent for patients with chronic heart failure.