- Email: [email protected]
Cardiomyocyte Apoptosis in Pressure Overloaded Myocardium: Possible Role of Calpain-Mediated Gelsolin Cleavage
The 11th Annual Scientific Meeting
HFSA
S97
079
081
Impaired Systolic Ventricular Function with Improved Myocardial Mechanics in Streptozotocin-Induced Diabetes Marina P. Okoshi1, Julliano F.C. Guimaraes1, Bruno P. Muzio1, Mario M. Sugizaki1, Andre F. Nascimento1, Ana A.H. Fernandes2, Maeli Dal Pai-Silva2, Antonio C. Cicogna1, Katashi Okoshi1; 1Internal Medicine, Faculdade de Medicina de Botucatu, UNESP, Botucatu, SP, Brazil; 2Morfologia, Instituto de Biociencias, UNESP, Botucatu, SP, Brazil
Cardiomyocyte Apoptosis in Pressure Overloaded Myocardium: Possible Role of Calpain-Mediated Gelsolin Cleavage Santosh K. Mani1, Hirokazu Shiraishi1, Sundaravadivel Balasubramanian1, Kentaro Yamane1, Meenakshi Chellaiah2, Sui X. Cai3, Janet M. Boggs1, George Cooper1, Michael R. Zile1, Dhandapani Kuppuswamy1; 1Medicine, Medical University of South Carolina and VA Medical Center, Charleston, SC; 2University of Maryland, Baltimore, MD; 3EpiCept Corporation, San Diego, CA
Diabetes mellitus results in cardiac dysfunction; however, the direct effects of diabetes on myocardial performance are controversial. This study examined in vivo ventricular function and in vitro myocardial performance in streptozotocin (STZ)induced diabetic rats. Methods: Male Wistar rats were intraperitoneally injected with STZ (60 mg/kg, n 5 16) or vehicle only (C, n 5 14). Thirty days later, left ventricular (LV) systolic performance was analysed by transthoracic echocardiography. Myocardial function was studied in isolated left ventricular papillary muscle under isometric contraction. Contractile reserve was assessed by increasing extracellular Ca2þ concentration from 1.25 mM to 5.20 mM, and by adding the beta-adrenergic agonist isoproterenol (1 mM) to the nutrient solution. Results: Hyperglycemia was detected within three days and remained elevated until the end of the study (C: 99 6 14; STZ: 398 6 57 mg/dL; p ! 0.001). LV fractional shortening (C: 54.1 6 5.2; STZ: 48.7 6 3.4; p ! 0.005) and LV posterior wall shortening velocity (C: 44.5 6 4.6; STZ: 35.5 6 5.4 mm/s; p ! 0.001) were decreased in the STZ group. Conclusion: The in vitro normal contractile reserve and improved myocardial function suggest that metabolic changes may be involved in the impaired in vivo systolic performance.
Background: Gelsolin cleavage causes actin disruption and apoptotic cell death. Activation of proteases such as caspase-3 and calpain has been shown to cleave gelsolin. We hypothesized that pressure-overload (PO) causes activation of proteases, gelsolin cleavage and cardiomyocyte apoptosis; furthermore, inhibition of protease activation will prevent gelsolin cleavage and apoptosis. Methods: Feline right ventricular PO was created using pulmonary artery banding for 48 hours, sham operated cats were used as controls (sham). Calpain activation, caspase-3 activation and gelsolin cleavage were measured by immunohistochemistry and Western blot. Apoptosis was measured by TUNEL staining and histone-2B phosphorylation. Results: Calpain was inhibited with calpeptin (two doses at 0.5 mg/kg), a peptide aldehydic inhibitor that binds irreversibly to the protease cleavage site of calpain; Caspase-3 was inhibited with MX1013 (20 mg/kg first dose and 10 mg/kg second dose), each drug was given by bolus intravenous injections twice, one 15 min before and the second 6 h after the induction of PO. Compared to sham (n 5 6), at 48 h post banding, PO (n 5 6) caused calpain and caspase-3 activation, gelsolin cleavage and increased cardiomyocyte apoptosis. Although administration of MX1013 (n 5 4) and calpeptin (n 5 4) blocked the activation of their respective proteases during PO, only calpeptin abolished cardiomyocyte apoptosis and gelsolin cleavage and enrichment in cardiomyocytes. Conclusion: Calpain activation in the PO myocardium leads to apoptosis via gelsolin cleavage. Calpain inhibition could be a potential therapeutic target for preserving myocardial structure and function by preventing cardiomyocyte apoptosis.
Papillary muscle data (mean 6 SD) 2
DT (g/mm ) 1.25 mM Ca2þ 5.20 mM Ca2þ 1 mM Isoproterenol
C
STZ
4.58 6 0.83 5.72 6 0.91 5.43 6 0.96
6.39 6 0.79* 7.03 6 0.87 6.70 6 0.66
DT: developed tension; *p ! 0.05 vs C; Student’s t test.
080 Rosiglitizone Improves Myocardial Insulin Sensitivity and Protects Against an Accelerated Course of Dilated Cardiomyopathy in Conscious, Chronically Instrumented Senescent Beagles Siva Bhashyam2, Suzanne B. Brown1, Pratik Parikh2, Brandy Patterson2, Richard P. Shannon1; 1Cardiology, Hospital of the University of Pennsylvania, Philadelphia, PA; 2 Medicine, Alleghany General Hospital, Pittsburgh, PA Introduction: Prior work from our laboratory has demonstrated that, when compared to younger dogs, senescent beagles have whole body and myocardial insulin resistance that predisposes to an accelerated course of dilated cardiomyopathy (DCM) following rapid pacing. These metabolic perturbations were observed despite controlling for weight and level of activity. Hypothesis: We sought to determine if the PPARg agonist Rosiglitizone (Rosi)would improve insulin sensitivity and protect against an accelerated course of DCM. Methods: Sixteen senescent (11-12 years) beagles underwent chronic instrumentation with LV pressure gauges, aortic and coronary sinus catheters to measure trans-myocardial substrate uptake, left circumflex coronary flow probe, and ultrasonic dimensions crystals to measure LV chamber dimensions. Six dogs were treated with Rosi (8 mg/day) for 8 weeks prior to instrumentation and 10 dogs served as age and weight matched controls (Con). Prior to treatment, all dogs underwent an IV glucose tolerance test (IVGGT). After treatment and prior to the initiation of rapid pacing (240 min-1), hyperinsulinemic-euglycemic clamps were performed to determine whole body and myocardial insulin sensitivity. Results: Consistent with our previous findings, both groups of senescent dogs had elevated NEFA (Con: 876 6 78; Rosi: 913 6 98 mmol/L) and insulin (Con: 112 6 27; Rosi: 101 6 36 pmol/L) prior to treatment, with comparable IVGTT. There were no significant differences in baseline hemodynamics. Rosi treatment resulted in reduced plasma NEFA (Con: 853 6 34; Rosi: 531 6 33mmol/L, p ! 0.02) and improved myocardial insulin responsiveness (Con: 289 6 54; Rosi: 512 6 44 mg/kg/100 mg, p ! 0.05). The increase in insulin sensitivity was associated with increased UCP-3 expression (Con: 226 6 41; Rosi: 512 6 33 DU, p ! 0.05) in isolated cardiomyocyte mitochondria. Rosiglitizone treatment increased the latency to the onset of DCM following rapid pacing (Con: 8 days; Rosi: 23 days, p ! 0.05) and reduced mortality (Con: 30%; Rosi: 0% at 28 days). Conclusion: Treatment of senescent beagles with Rosiglitizone improves whole body and myocardial insulin sensitivity, increases myocardial UCP-3 expression, and protects against an accelerated course of DCM and premature mortality.
HFSA
S97
079
081
Impaired Systolic Ventricular Function with Improved Myocardial Mechanics in Streptozotocin-Induced Diabetes Marina P. Okoshi1, Julliano F.C. Guimaraes1, Bruno P. Muzio1, Mario M. Sugizaki1, Andre F. Nascimento1, Ana A.H. Fernandes2, Maeli Dal Pai-Silva2, Antonio C. Cicogna1, Katashi Okoshi1; 1Internal Medicine, Faculdade de Medicina de Botucatu, UNESP, Botucatu, SP, Brazil; 2Morfologia, Instituto de Biociencias, UNESP, Botucatu, SP, Brazil
Cardiomyocyte Apoptosis in Pressure Overloaded Myocardium: Possible Role of Calpain-Mediated Gelsolin Cleavage Santosh K. Mani1, Hirokazu Shiraishi1, Sundaravadivel Balasubramanian1, Kentaro Yamane1, Meenakshi Chellaiah2, Sui X. Cai3, Janet M. Boggs1, George Cooper1, Michael R. Zile1, Dhandapani Kuppuswamy1; 1Medicine, Medical University of South Carolina and VA Medical Center, Charleston, SC; 2University of Maryland, Baltimore, MD; 3EpiCept Corporation, San Diego, CA
Diabetes mellitus results in cardiac dysfunction; however, the direct effects of diabetes on myocardial performance are controversial. This study examined in vivo ventricular function and in vitro myocardial performance in streptozotocin (STZ)induced diabetic rats. Methods: Male Wistar rats were intraperitoneally injected with STZ (60 mg/kg, n 5 16) or vehicle only (C, n 5 14). Thirty days later, left ventricular (LV) systolic performance was analysed by transthoracic echocardiography. Myocardial function was studied in isolated left ventricular papillary muscle under isometric contraction. Contractile reserve was assessed by increasing extracellular Ca2þ concentration from 1.25 mM to 5.20 mM, and by adding the beta-adrenergic agonist isoproterenol (1 mM) to the nutrient solution. Results: Hyperglycemia was detected within three days and remained elevated until the end of the study (C: 99 6 14; STZ: 398 6 57 mg/dL; p ! 0.001). LV fractional shortening (C: 54.1 6 5.2; STZ: 48.7 6 3.4; p ! 0.005) and LV posterior wall shortening velocity (C: 44.5 6 4.6; STZ: 35.5 6 5.4 mm/s; p ! 0.001) were decreased in the STZ group. Conclusion: The in vitro normal contractile reserve and improved myocardial function suggest that metabolic changes may be involved in the impaired in vivo systolic performance.
Background: Gelsolin cleavage causes actin disruption and apoptotic cell death. Activation of proteases such as caspase-3 and calpain has been shown to cleave gelsolin. We hypothesized that pressure-overload (PO) causes activation of proteases, gelsolin cleavage and cardiomyocyte apoptosis; furthermore, inhibition of protease activation will prevent gelsolin cleavage and apoptosis. Methods: Feline right ventricular PO was created using pulmonary artery banding for 48 hours, sham operated cats were used as controls (sham). Calpain activation, caspase-3 activation and gelsolin cleavage were measured by immunohistochemistry and Western blot. Apoptosis was measured by TUNEL staining and histone-2B phosphorylation. Results: Calpain was inhibited with calpeptin (two doses at 0.5 mg/kg), a peptide aldehydic inhibitor that binds irreversibly to the protease cleavage site of calpain; Caspase-3 was inhibited with MX1013 (20 mg/kg first dose and 10 mg/kg second dose), each drug was given by bolus intravenous injections twice, one 15 min before and the second 6 h after the induction of PO. Compared to sham (n 5 6), at 48 h post banding, PO (n 5 6) caused calpain and caspase-3 activation, gelsolin cleavage and increased cardiomyocyte apoptosis. Although administration of MX1013 (n 5 4) and calpeptin (n 5 4) blocked the activation of their respective proteases during PO, only calpeptin abolished cardiomyocyte apoptosis and gelsolin cleavage and enrichment in cardiomyocytes. Conclusion: Calpain activation in the PO myocardium leads to apoptosis via gelsolin cleavage. Calpain inhibition could be a potential therapeutic target for preserving myocardial structure and function by preventing cardiomyocyte apoptosis.
Papillary muscle data (mean 6 SD) 2
DT (g/mm ) 1.25 mM Ca2þ 5.20 mM Ca2þ 1 mM Isoproterenol
C
STZ
4.58 6 0.83 5.72 6 0.91 5.43 6 0.96
6.39 6 0.79* 7.03 6 0.87 6.70 6 0.66
DT: developed tension; *p ! 0.05 vs C; Student’s t test.
080 Rosiglitizone Improves Myocardial Insulin Sensitivity and Protects Against an Accelerated Course of Dilated Cardiomyopathy in Conscious, Chronically Instrumented Senescent Beagles Siva Bhashyam2, Suzanne B. Brown1, Pratik Parikh2, Brandy Patterson2, Richard P. Shannon1; 1Cardiology, Hospital of the University of Pennsylvania, Philadelphia, PA; 2 Medicine, Alleghany General Hospital, Pittsburgh, PA Introduction: Prior work from our laboratory has demonstrated that, when compared to younger dogs, senescent beagles have whole body and myocardial insulin resistance that predisposes to an accelerated course of dilated cardiomyopathy (DCM) following rapid pacing. These metabolic perturbations were observed despite controlling for weight and level of activity. Hypothesis: We sought to determine if the PPARg agonist Rosiglitizone (Rosi)would improve insulin sensitivity and protect against an accelerated course of DCM. Methods: Sixteen senescent (11-12 years) beagles underwent chronic instrumentation with LV pressure gauges, aortic and coronary sinus catheters to measure trans-myocardial substrate uptake, left circumflex coronary flow probe, and ultrasonic dimensions crystals to measure LV chamber dimensions. Six dogs were treated with Rosi (8 mg/day) for 8 weeks prior to instrumentation and 10 dogs served as age and weight matched controls (Con). Prior to treatment, all dogs underwent an IV glucose tolerance test (IVGGT). After treatment and prior to the initiation of rapid pacing (240 min-1), hyperinsulinemic-euglycemic clamps were performed to determine whole body and myocardial insulin sensitivity. Results: Consistent with our previous findings, both groups of senescent dogs had elevated NEFA (Con: 876 6 78; Rosi: 913 6 98 mmol/L) and insulin (Con: 112 6 27; Rosi: 101 6 36 pmol/L) prior to treatment, with comparable IVGTT. There were no significant differences in baseline hemodynamics. Rosi treatment resulted in reduced plasma NEFA (Con: 853 6 34; Rosi: 531 6 33mmol/L, p ! 0.02) and improved myocardial insulin responsiveness (Con: 289 6 54; Rosi: 512 6 44 mg/kg/100 mg, p ! 0.05). The increase in insulin sensitivity was associated with increased UCP-3 expression (Con: 226 6 41; Rosi: 512 6 33 DU, p ! 0.05) in isolated cardiomyocyte mitochondria. Rosiglitizone treatment increased the latency to the onset of DCM following rapid pacing (Con: 8 days; Rosi: 23 days, p ! 0.05) and reduced mortality (Con: 30%; Rosi: 0% at 28 days). Conclusion: Treatment of senescent beagles with Rosiglitizone improves whole body and myocardial insulin sensitivity, increases myocardial UCP-3 expression, and protects against an accelerated course of DCM and premature mortality.