Ricky Malhotra

Glucose Uptake and Glycolysis Reduce Hypoxia-induced Apoptosis in Cultured Neonatal Rat Cardiac Myocytes

Myocardial ischemia/reperfusion is well recognized as a major cause of apoptotic or necrotic cell death. Neonatal rat cardiac myocytes are intrinsically resistant to hypoxia-induced apoptosis, suggesting a protective role of energy-generating substrates. In the present report, a model of sustained hypoxia of primary cultures of Percoll-enriched neonatal rat cardiac myocytes was used to study specifically the modulatory role of extracellular glucose and other intermediary substrates of energy metabolism (pyruvate, lactate, propionate) as well as glycolytic inhibitors (2-deoxyglucose and iodoacetate) on the induction and maintenance of apoptosis. In the absence of glucose and other substrates, hypoxia (5% CO2 and 95% N2) caused apoptosis in 14% of cardiac myocytes at 3 h and in 22% of cells at 6–8 h of hypoxia, as revealed by sarcolemmal membrane blebbing, nuclear fragmentation, and chromatin condensation (Hoechst staining), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and DNA laddering. This was accompanied by translocation of cytochrome c from the mitochondria to the cytosol and cleavage of the death substrate poly(ADP-ribose) polymerase. Cleavage of poly(ADP-ribose) polymerase and DNA laddering were prevented by preincubation with the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (zDEVD-fmk), indicating activation of caspases in the apoptotic process. The caspase inhibitor zDEVD-fmk also partially inhibited cytochrome c translocation. The presence of as little as 1 mm glucose, but not pyruvate, lactate, or propionate, before hypoxia prevented apoptosis. Inhibiting glycolysis by 2-deoxyglucose or iodoacetate, in the presence of glucose, reversed the protective effect of glucose. This study demonstrates that glycolysis of extracellular glucose, and not other metabolic pathways, protects cardiac myocytes from hypoxic injury and subsequent apoptosis.

Mechanical Stretch and Angiotensin II Differentially Upregulate the Renin-Angiotensin System in Cardiac Myocytes In Vitro

Pressure overload in vivo results in left ventricular hypertrophy and activation of the renin-angiotensin system in the heart. Mechanical stretch of neonatal rat cardiac myocytes in vitro causes secretion of angiotensin II (Ang II), which in turn plays a pivotal role in mechanical stretch-induced hypertrophy. Although in vivo data suggest that the stimulus of hemodynamic overload serves as an important modulator of cardiac renin-angiotensin system (RAS) activity, it is not clear whether observed upregulation of RAS genes is a direct effect of hemodynamic stress or is secondary to neurohumoral effects in response to hemodynamic overload. Moreover, it is unclear whether activation of the local RAS in response to hemodynamic overload predominantly occurs in cardiac myocytes or fibroblasts or both. In the present study, we examined the effect of mechanical stretch on expression of angiotensinogen, renin, angiotensin-converting enzyme (ACE), and Ang II receptor (AT(1A), AT(1B), and AT(2)) genes in neonatal rat cardiac myocytes and cardiac fibroblasts in vitro. The level of expression of angiotensinogen, renin, ACE, and AT(1A) genes was low in unstretched cardiac myocytes, but stretch upregulated expression of these genes at 8 to 24 hours. Stimulation of cardiac myocytes with Ang II also upregulated expression of angiotensinogen, renin, and ACE genes, whereas it downregulated AT(1A) and did not affect AT(1B) gene expression. Although losartan, a specific AT(1) antagonist, completely inhibited Ang II-induced upregulation of angiotensinogen, renin, and ACE genes, as well as stretch-induced upregulation of AT(1A) expression, it did not block upregulation of angiotensinogen, renin, and ACE genes by stretch. Western blot analyses showed increased expression of angiotensinogen and renin protein at 16 to 24 hours of stretch. The ACE-like activity was also significantly elevated at 24 hours after stretch. Radioligand binding assays revealed that stretch significantly upregulated the AT(1) density on cardiac myocytes. Interestingly, stretch of cardiac fibroblasts did not result in any discernible increases in the expression of RAS genes. Our results indicate that mechanical stretch in vitro upregulates both mRNA and protein expression of RAS components specifically in cardiac myocytes. Furthermore, components of the cardiac RAS are independently and differentially regulated by mechanical stretch and Ang II in neonatal rat cardiac myocytes.

Hypoxia-inducible factor-1alpha is a critical mediator of hypoxia induced apoptosis in cardiac H9c2 and kidney epithelial HK-2 cells

BackgroundHypoxia inducible factor-1 (HIF-1) is a transcription factor that functions to maintain cellular homeostasis in response to hypoxia. There is evidence that HIF-1 can also trigger apoptosis, possibly when cellular responses are inadequate to meet energy demands under hypoxic conditions.MethodsCardiac derived H9c2 and renal tubular epithelial HK-2 cells expressing either the wild type oxygen regulated subunit of HIF-1 (pcDNA3-Hif-1α) or a dominant negative version that lacked both DNA binding and transactivation domains (pcDNA3-DN-Hif-1α), were maintained in culture and exposed to hypoxia. An RNA interference approach was also employed to selectively knockdown expression of Hif-1α. Apoptosis was analyzed in both H9c2 and HK-2 cells by Hoechst and TUNEL staining, caspase 3 activity assays and activation of pro-apoptotic Bcl2 family member Bax.ResultsOverexpression of pcDNA3-DN-Hif-1α led to a significant reduction in hypoxia -induced apoptosis (17 ± 2%, P < 0.01) in H9c2 cells compared to both control-transfected and wild type Hif-1α transfected cells. Moreover, selective ablation of HIF-1α protein expression by RNA interference in H9c2 cells led to 55% reduction of caspase 3 activity and 46% reduction in the number of apoptotic cells as determined by Hoechst 33258 staining, after hypoxia. Finally, upregulation of the pro-apoptotic protein, Bax, was found in H9c2 cells overexpressing full-length pcDNA3-HA-HIF-1α exposed to hypoxia. In HK-2 cells overexpression of wild-type Hif-1α led to a two-fold increase in Hif-1α levels during hypoxia. This resulted in a 3.4-fold increase in apoptotic cells and a concomitant increase in caspase 3 activity during hypoxia when compared to vector transfected control cells. HIF-1α also induced upregulation of Bax in HK-2 cells. In addition, introduction of dominant negative Hif-1α constructs in both H9c2 and HK-2 -cells led to decreased active Bax expression.ConclusionThese data demonstrate that HIF-1α is an important component of the apoptotic signaling machinery in the two cell types.

Gαq-mediated activation of GRK2 by mechanical stretch in cardiac myocytes. THE ROLE OF PROTEIN KINASE C.

G protein-coupled receptor kinase-2 (GRK2) is a critical regulator of β-adrenergic receptor (β-AR) signaling and cardiac function. We studied the effects of mechanical stretch, a potent stimulus for cardiac myocyte hypertrophy, on GRK2 activity and β-AR signaling. To eliminate neurohormonal influences, neonatal rat ventricular myocytes were subjected to cyclical equi-biaxial stretch. A hypertrophic response was confirmed by “fetal” gene up-regulation. GRK2 activity in cardiac myocytes was increased 4.2-fold at 48 h of stretch versus unstretched controls. Adenylyl cyclase activity was blunted in sarcolemmal membranes after stretch, demonstrating β-AR desensitization. The hypertrophic response to mechanical stretch is mediated primarily through the Gαq-coupled angiotensin II AT1 receptor leading to activation of protein kinase C (PKC). PKC is known to phosphorylate GRK2 at the N-terminal serine 29 residue, leading to kinase activation. Overexpression of a mini-gene that inhibits receptor-Gαq coupling blunted stretch-induced hypertrophy and GRK2 activation. Short hairpin RNA-mediated knockdown of PKCα also significantly attenuated stretch-induced GRK2 activation. Overexpression of a GRK2 mutant (S29A) in cardiac myocytes inhibited phosphorylation of GRK2 by PKC, abolished stretch-induced GRK2 activation, and restored adenylyl cyclase activity. Cardiac-specific activation of PKCα in transgenic mice led to impaired β-agonist-stimulated ventricular function, blunted cyclase activity, and increased GRK2 phosphorylation and activity. Phosphorylation of GRK2 by PKC appears to be the primary mechanism of increased GRK2 activity and impaired β-AR signaling after mechanical stretch. Cross-talk between hypertrophic signaling at the level of PKC and β-AR signaling regulated by GRK2 may be an important mechanism in the transition from compensatory ventricular hypertrophy to heart failure.

G Protein-coupled Receptor Kinase-2 Is a Novel Regulator of Collagen Synthesis in Adult Human Cardiac Fibroblasts

G protein-coupled receptor kinase-2 is a novel regulator of collagen synthesis in adult human cardiac fibroblasts. Karen M. D’Souza, Ricky Malhotra, Jennifer L. Philip, Michelle L. Staron, Tiju Theccanat, Valluvan Jeevanandam, and Shahab A. Akhter This article has been withdrawn by the authors. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 43, p. 25849, October 23, 2015

Reversal of impaired myocardial β-adrenergic receptor signaling by continuous-flow left ventricular assist device support

BACKGROUND Myocardial beta-adrenergic receptor (beta-AR) signaling is severely impaired in chronic heart failure (HF). This study was conducted to determine if left ventricular (LV) beta-AR signaling could be restored after continuous-flow LV assist device (LVAD) support. METHODS Twelve patients received LVADs as a bridge to transplant. Paired LV biopsy specimens were obtained at the time of LVAD implant (HF group) and transplant (LVAD group). The mean duration of LVAD support was 152 +/- 34 days. Myocardial beta-AR signaling was assessed by measuring adenylyl cyclase (AC) activity, total beta-AR density (B(max)), and G protein-coupled receptor kinase-2 (GRK2) expression and activity. LV specimens from 8 non-failing hearts (NF) were used as controls. RESULTS Basal and isoproterenol-stimulated AC activity was significantly lower in HF vs NF, indicative of beta-AR uncoupling. Continuous-flow LVAD support restored basal and isoproterenol-stimulated AC activity to levels similar to NF. B(max) was decreased in HF vs NF and increased to nearly normal in the LVAD group. GRK2 expression was increased 2.6-fold in HF vs NF and was similar to NF after LVAD support. GRK2 activity was 3.2-fold greater in HF vs NF and decreased to NF levels in the LVAD group. CONCLUSIONS Myocardial beta-AR signaling can be restored to nearly normal after continuous-flow LVAD support. This is similar to previous data for volume-displacement pulsatile LVADs. Decreased GRK2 activity is an important mechanism and indicates that normalization of the neurohormonal milieu associated with HF is similar between continuous-flow and pulsatile LVADs. This may have important implications for myocardial recovery.

The role of the cardiac renin-angiotensin system inload-induced cardiac hypertrophy

Hypertrophy is a fundamental adaptive process employed by postmitotic cardiac and skeletal muscles in response to mechanical load. How muscle cells convert mechanical stimuli into growth signals has been a long-standing question. Using an in vitro model of stretch-induced cardiac hypertrophy, we and others have demonstrated that mechanical stretch causes secretion of angiotensin II from cardiac myocytes and this acts as a critical mediator of the stretch-induced hypertrophic response. The results provide direct evidence for an autocrine mechanism in load-induced growth of cardiac muscle cells in vitro. In this brief review, we discuss the role of the cardiac renin-angiotensin system in load-induced cardiac hypertrophy and describe our current hypothesis and unsolved questions on the mechanism of stretch-induced hypertrophy in the neonatal rat cardiac myocyte system.

High-molecular-weight polyethylene glycol protects cardiac myocytes from hypoxia- and reoxygenation-induced cell death and preserves ventricular function

Apoptosis plays a significant role in maladaptive remodeling and ventricular dysfunction following ischemia-reperfusion injury. There is a critical need for novel approaches to inhibit apoptotic cell death following reperfusion, as this loss of cardiac myocytes can progressively lead to heart failure. We investigated the ability and signaling mechanisms of a high-molecular-weight polyethylene glycol-based copolymer, PEG 15-20, to protect cardiac myocytes from hypoxia-reoxygenation (H-R)-induced cell death and its efficacy in preserving ventricular function following extended hypothermic ischemia and warm reperfusion as relevant to cardiac transplantation. Pretreatment of neonatal rat ventricular myocytes with a 5% PEG solution led to a threefold decline in apoptosis after H-R relative to untreated controls. There was a similar decline in caspase-3 activity in conjunction with inhibition of cytochrome c release from the inner mitochondrial membrane. Treatment with PEG also reduced reactive oxygen species production after H-R, and sarcolemmal lipid-raft architecture was preserved, consistent with membrane stabilization. Cell survival signaling was upregulated after H-R with PEG, as demonstrated by increased phosphorylation of Akt, GSK-3β, and ERK1/2. There was also maintenance of cardiac myocyte β-adrenergic signaling, which is critical for myocardial function. PEG 15-20 was very effective in preserving left ventricular function following prolonged hypothermic ischemia and warm reperfusion. PEG 15-20 has a potent protective antiapoptotic effect in cardiac myocytes exposed to H-R injury and may represent a novel therapeutic strategy to decrease myocardial cell death and ventricular dysfunction at the time of reperfusion during acute coronary syndrome or following prolonged donor heart preservation.

Activation of JAK-STAT and nitric oxide signaling as a mechanism for donor heart dysfunction

BACKGROUND Donor heart dysfunction (DHD) precluding procurement for transplantation occurs in up to 25% of brain-dead (BD) donors. The molecular mechanisms of DHD remain unclear. We investigated the potential role of myocardial interleukin (IL)-6 signaling through the JAK2-STAT3 pathway, which can lead to the generation of nitric oxide (NO) and decreased cardiac myocyte contractility. METHODS Hearts were procured using standard technique with University of Wisconsin (UW) solution from 14 donors with a left ventricular (LV) ejection fraction of <35% (DHD). Ten hearts with normal function (NF) after BD served as controls. LV IL-6 was quantitated by enzyme-linked immunoassay (ELISA) and JAK2-STAT3 signaling was assessed by expression of phosphorylated STAT3. Inducible NO synthase (iNOS) and caspase-3 were measured by activity assays. RESULTS Myocardial IL-6 expression was 8-fold greater in the DHD group vs NF controls. Phosphorylated STAT3 expression was 5-fold higher in DHD than in NF, indicating increased JAK2-STAT3 signaling. LV activity of iNOS was 2.5-fold greater in DHD than in NF. LV expression of the pro-apoptotic gene Bnip3 and caspase-3 activity were 3-fold greater in the DHD group than in the NF group. CONCLUSIONS Myocardial IL-6 expression is significantly higher in the setting of DHD compared with hearts procured with normal function. This may lead to increased JAK2-STAT3 signaling and upregulation of iNOS, which has been shown to decrease cardiac myocyte contractility. Increased NO production may also lead to increased apoptosis through upregulation of Bnip3 gene expression. Increased iNOS signaling may be an important mechanism of DHD and represents a novel therapeutic target to improve cardiac function after BD.

Mechanical Stress, Local Renin-Angiotensin System and Cardiac Hypertrophy: An Overview

Hypertrophy is a fundamental adaptive process employed by postmitotic cardiac and skeletal muscles in response to mechanical load. External load also plays a critical role in determining muscle mass and its phenotype in cardiac myocytes. Interestingly, cardiac myocytes have the intrinsic ability to sense mechanical stretch and convert it into intracellular growth signals, which finally culminate in hypertrophic growth. Mechanical stretch of cardiac myocytes in vitro causes activation of multiple messenger systems, upregulation of many immediate early genes (e.g., c-fos, c-myc, c-jun, etc.), and re-expression of fetal-type genes (e.g., atrial natriuretic factor, skeletal a-actin, β-myosin heavy chain), reminiscent of cardiac hypertrophy in vivo. Stretch of neonatal rat cardiac myocytes stimulates a rapid secretion of angiotensin (Ang) II and an upregulation of all major components of cardiac renin-angiotensin system (RAS) genes. Ang II, along with other (secreted) growth factors, mediates many, if not all, stretch-induced hypertrophic responses. In this review, the relationship between mechanical loading, cardiac RAS, and cardiac hypertrophy is discussed. In addition, various cell signaling mechanisms initiated by mechanical stress on cardiac myocytes are briefly summarized.