Search

Right ventricular (RV) failure is the major determinant of outcome in pulmonary hypertension (PH). Calves exposed to 2-wk hypoxia develop severe PH and unlike rodents, hypoxia-induced PH in this species can lead to right heart failure. We, therefore, sought to examine the molecular and structural changes in the RV in calves with hypoxia-induced PH, hypothesizing that we could identify mechanisms underlying compensated physiological function in the face of developing severe PH. Calves were exposed to 14 days of environmental hypoxia (equivalent to 4,570 m/15,000 ft elevation, n = 29) or ambient normoxia (1,525 m/5,000 ft, n = 25). Cardiopulmonary function was evaluated by right heart catheterization and pressure volume loops. Molecular and cellular determinants of RV remodeling were analyzed by cDNA microarrays, RealTime PCR, proteomics, and immunochemistry. Hypoxic exposure induced robust PH, with increased RV contractile performance and preserved cardiac output, yet evidence of dysregulated RV-pulmonary artery mechanical coupling as seen in advanced disease. Analysis of gene expression revealed cellular processes associated with structural remodeling, cell signaling, and survival. We further identified specific clusters of gene expression associated with 1) hypertrophic gene expression and prosurvival mechanotransduction through YAP-TAZ signaling, 2) extracellular matrix (ECM) remodeling, 3) inflammatory cell activation, and 4) angiogenesis. A potential transcriptomic signature of cardiac fibroblasts in RV remodeling was detected, enriched in functions related to cell movement, tissue differentiation, and angiogenesis. Proteomic and immunohistochemical analysis confirmed RV myocyte hypertrophy, together with localization of ECM remodeling, inflammatory cell activation, and endothelial cell proliferation within the RV interstitium. In conclusion, hypoxia and hemodynamic load initiate coordinated processes of protective and compensatory RV remodeling to withstand the progression of PH. NEW & NOTEWORTHY Using a large animal model and employing a comprehensive approach integrating hemodynamic, transcriptomic, proteomic, and immunohistochemical analyses, we examined the early (2 wk) effects of severe PH on the RV. We observed that RV remodeling during PH progression represents a continuum of transcriptionally driven processes whereby cardiac myocytes, fibroblasts, endothelial cells, and proremodeling macrophages act to coordinately maintain physiological homeostasis and protect myocyte survival during chronic, severe, and progressive pressure overload. [ABSTRACT FROM AUTHOR]

Adenosine signaling has been implicated in cardiac adaptation to limited oxygen availability. In a wide search for adenosine receptor A2b (Adora2b)-elicited cardioadaptive responses, we identified the circadian rhythm protein period 2 (Per2) as an Adora2b target. Adora2b signaling led to Per2 stabilization during myocardial ischemia, and in this setting, [Per2.sup.-/-] mice had larger infarct sizes compared to wild-type mice and loss of the cardioprotection conferred by ischemic preconditioning. Metabolic studies uncovered a limited ability of ischemic hearts in [Per2.sup.-/-] mice to use carbohydrates for oxygen-efficient glycolysis. This impairment was caused by a failure to stabilize hypoxia-inducible factor-1a (Hif-1a). Moreover, stabilization of Per2 in the heart by exposing mice to intense light resulted in the transcriptional induction of glycolytic enzymes and Per2-dependent cardioprotection from ischemia. Together, these studies identify adenosine-elicited stabilization of Per2 in the control of HIF-dependent cardiac metabolism and ischemia tolerance and implicate Per2 stabilization as a potential new strategy for treating myocardial ischemia., Metabolic adaptation during environmental stress is currently an area of intense investigation because of the potential implications of metabolic alterations in human disease (1), (2). For example, myocardial ischemia leads [...]

This study was performed to elucidate the relation between in viva measurements of two-dimensional principal strains and the progression of left ventricle (LV) wall thinning during development of dilated cardiomyopathy in the protein kinase C-[epsilon] (PKC-[epsilon]) transgenic (TG) overexpressing mouse heart. Principal two-dimensional strains, E1 and E2, were determined in the LV wall of the anesthetized mouse using cardiac MRI tagging at 14.1 T. PKC-[epsilon] TG provided a model of pure dilated cardiomyopathy without evidence of hypertrophy (PKC-[epsilon] TG, n = 6). Ejection fraction, wall thickness, and principal strains were determined at 1-mo intervals in hearts of PKC-[epsilon] TG vs. age-matched, nontransgenic mice (NTG, n = 5) from age 6 to 13 mo. Through the study, PKC-[epsilon] TG displayed lower ejection fraction than NTG. At 7 mo, average principal strain E1 in PKC-[epsilon] TG hearts was lower compared with NTG (PKC-[espilon] TG = 0.14 [+ or -] 0.03, NTG = 0.19 [+ or -] 0.03, P < 0.05). The greatest reductions in regional E1 occurred in the lateral segments. The principal strain E2 did not change significantly in either group. At 9 mo, LV wall thinning occurred in PKC-[epsilon] TG mice (P < 0.01 vs. 8 mo) to 21% below values in NTG (P < 0.001). Average E1 strain diverged between PKC-[epsilon] TG and NTG hearts by 25-43%. These E1 changes preceded LV wall thinning and predated the eventual transition from a compensated circumstance to the dilated phenotype. The findings indicate a near step function in E1 depression that precedes the onset of LV wall thinning and suggest El as a prognostic indicator of dilated cardiomyopathy. protein kinase C; magnetic resonance imaging; ventricular function

The cardiac myosin binding protein C (cMyBPC) phosphorylation in [PKC.sub.E] TG mice is investigated. It is found that the Ser302 on mouse cMyBPC is a likely [PKC.sub.E] phosphorylation site in vivo and in vitro.

Glucose metabolism in the heart requires oxidation of cytosolic NADH from glycolysis. This study examines shuttling reducing equivalents from the cytosol to the mitochondria via the activity and expression of the oxoglutarate-malate carrier (OMC) in rat hearts subjected to 2 wk (Hyp2, n = 6) and 10 wk (Hyp10, n = 8) of pressure overload hypertrophy vs. that of sham-operated rats (Sham2, n = 6; and Sham10, n = 7). Moderate aortic banding produced increased atrial natriuretic factor (ANF) mRNA expression at 2 and 10 wk, but only at 10 wk did hearts develop compensatory hypertrophy (33% increase, P < 0.05). Isolated hearts were perfused with the short-chain fatty acid [2,4-[sup.13][C.sub.2]]butyrate (2 mM) and glucose (5 nM) to enable dynamic-mode [sup.13]C NMR of intermediate exchange across OMC. OMC flux increased before the development of hypertrophy: Hyp2 = 9.6 [+ or -] 2.1 vs. Sham2 = 3.7 [+ or -] 1.2 [micro]M x [min.sup.-1] x g dry [wt.sup.-1], providing an increased contribution of cytosolic NADH to energy synthesis in the mitochondria. With compensatory hypertrophy, OMC flux returned to normal: Hyp10 = 3.9 [+ or -] 1.7 vs. Sham10 = 3.8 [+ or -] 1.2 [micro]M x [g.sup.-1] x [min.sup.-1]. Despite changes in activity, no differences in OMC expression occurred between Hyp and Sham groups. Elevated OMC flux represented augmented cytosolic NADH shuttling, coupled to increased nonoxidative glycolysis, in response to hypertrophic stimulus. However, development of compensatory hypertrophy moderated the pressure-induced elevation in OMC flux, which returned to control levels. The findings indicate that the challenge of pressure overload increases cytosolic redox state and its contribution to mitochondrial oxidation but that hypertrophy, before decompensation, alleviates this stress response. malate-aspartate shuttle; redox state; hypertrophy doi:10.1152/ajpcell.00576.2006

Hypoxia-inducible factor-1[alpha] (HIF-1[alpha]) plays a role in a number of cell-protective pathways after ischemia. There are clear sex-related differences in the remodeling process, and hearts from males tend to dilate in response to pathological loads and ischemia to a greater degree than do hearts from females. Thus we hypothesized that there would be a sex-related dimorphic response of HIF-1[alpha] to an ischemic event. Male and female rats were euthanized 5 and 24 h after coronary ligation (M-MI and F-MI; MI, myocardial ischemia), and HIF-1[alpha] expression was determined by immunohistochemistry, Western blot, and quantitative RT-PCR. Sham-operated male and female animals served as controls (M-SH and F-SH). In the ischemic area, histochemical analysis at 5 h showed that HIF was expressed in 33% of cell nuclei in M-MI and in 55% in F-MI. At 24 h, HIF expression increased to 49% in M-MI and to 82% in F-MI (P < 0.05 vs. SH and also M-MI vs. F-MI). This difference was not only statistically significant between the two sexes at 24 h but also within each sex at 5 and 24 h after ligation. Western blots confirmed that, at 24 h after ischemia, HIF protein increased significantly in both male and female hearts relative to sham-operated animals but that the increase in females was 60% greater than that seen in males, mRNA expression of HIF was significantly increased at 24 h in F-MI versus M-MI and sham-operated animals. Expression of downstream HIF target genes (heme oxygenase and brain natriuretic peptide) was increased in proportion to the levels of HIF expression. These data suggest a novel cellular mechanism to explain the sex-related dimorphic response to ischemia and also the possibility that exogenous modulation of HIF might represent a new therapeutic approach to preventing left ventricular remodeling. hypoxia-inducible factor-la; gene expression; remodeling doi:10.1152/ajpheart.00580.2005

Diabetes mellitus is associated with a distinct cardiomyopathy. Whether cardiac myofilament function is altered in human diabetes mellitus is unknown. Myocardial biopsies were obtained from seven diabetic patients and five control, nondiabetic patients undergoing coronary artery bypass surgery. Myofilament function was assessed by determination of the developed force-[Ca.sup.2+] concentration relation in skinned cardiac cells from flashfrozen human biopsies. Separate control experiments revealed that flash freezing of biopsy specimens did not affect myofilament function. All patients in the diabetes mellitus cohort were classified as Type 2 diabetes mellitus patients, and most showed signs of diastolic dysfunction. Diabetes mellitus was associated with depressed myofilament function, that is, decreased [Ca.sup.2+] sensitivity (29%, P < 0.05 vs. control) and a trend toward reduction of maximum [Ca.sup.2+]-saturated force (29%, P = 0.08 vs. control). The slope of the force-[Ca.sup.2+] concentration relation (Hill coefficient) was not affected by diabetes, however. We conclude that human diabetes mellitus is associated with decreased cardiac myofilament function. Depressed cardiac myofilament [Ca.sup.2+] responsiveness may underlie the decreased ventricular function characteristic of human diabetic cardiomyopathy. heart failure; myofilament function; skinned myocardium; sarcomere

Myofilament dysfunction is a common point of convergence for many forms of heart failure. Recently, we showed that cardiac overexpression of PKC[epsilon] initially depresses myofilament activity and then leads to a progression of changes characteristic of human heart failure. Here, we examined the effects of PKC[epsilon] on contractile reserve, Starling mechanism, and myofilament activation in this model of end-stage dilated cardiomyopathy. Pressure-volume loop analysis and echocardiography showed that the PKC[epsilon] mice have markedly compromised systolic function and increased end-diastolic volumes. Dobutamine challenge resulted in a small increase in contractility in PKC[epsilon] mice but failed to enhance cardiac output. The PKC[epsilon] mice showed a normal length-dependent tension development in skinned cardiac muscle preparations, although Frank-Starling mechanism appeared to be compromised in the intact animal. Simultaneous measurement of tension and ATPase demonstrated that the maximum tension and ATPase were markedly lower in the PKC[epsilon] mice at any length or [Ca.sup.2+] concentration. However, the tension cost was also lower indicating less energy expenditure. We conclude 1) that prolonged overexpression of PKC[epsilon] ultimately leads to a dilated cardiomyopathy marked by exhausted contractile reserve, 2) that PKC[epsilon] does not compromise the Frank-Starling mechanism at the myofilament level, and 3) that the Starling curve excursion is limited by the inotropic state of the heart. These results reflect the significance of the primary myofilament contractilopathy induced by phosphorylation and imply a role for PKC[epsilon]-mediated phosphorylation in myofilament physiology and the pathophysiology of decompensated cardiac failure. pressure-volume loops; cardiac myofilaments

Cardiac disease in diabetes presents as impaired left ventricular contraction and relaxation; however, the mechanisms underlying contractile protein dysfunction during the progression of disease are unknown. Accordingly, we assessed [Ca.sup.2+]-dependent tension development and tension-dependent ATP consumption (tension cost) in a rat model early (6 wk) and late (12 wk) after the onset of diabetes (50 mg/kg iv streptozotocin) using mechanical force- and enzyme-coupled UV absorbance measurements. Myofilament [Ca.sup.2+] sensitivity and maximal tension were unchanged between groups at either time point. Cross-bridge cycling rate was significantly decreased in diabetes, as indexed by tension cost (early control 5.4 [+ or -] 0.4 and early diabetes 4.2 [+ or -] 0.3; and late control 6.0 [+ or -] 0.2 and late diabetes 4.2 [+ or -] 0.2; P < 0.05). Because rodent models of cardiac disease are confounded by altered myosin isoform distribution, myosin content was determined by SDS-PAGE and densitometry. The cardiac content of [alpha]-myosin in diabetes was decreased to 41% [+ or -] 4.1 at 6 wk and 32.5% [+ or -] 2.9 at 12 wk of diabetes (early control 77.8% [+ or -] 3.3 and late control 73.6% [+ or -] 2.5). Separate control experiments demonstrated a linear decrease in tension cost with decreased [alpha]-myosin content. Given this, the depression of tension cost in this rodent model of diabetes could be fully explained by the altered myosin isoform distribution. cross-bridge cycling; cardiac energetics

Inhibition of PKC phosphorylation of cTnI improves cardiac performance in vivo. Am J Physiol Hert Circ Physiol 286: H2089-H2095, 2004. First published January 15, 2004; 10.1152/ajpbeart.00582.2003.--Protein kinase C (PKC) modulates cardiomyocyte function by phosphorylation of intracellular targets including myofilament proteins. Data generated from studies on in vitro heart preparations indicate that PKC phosphorylation of troponin I (TnI), primarily via PKC-[member of], may slow the rates of cardiac contraction and relaxation (+dP/dt and -dP/dt). To explore this issue in vivo, we employed transgenic mice [mutant TnI (mTnI) mice] in which the major PKC phosphorylation sites on cardiac TnI were mutated by alanine substitutions for [Ser.sup.43] and [Ser.sup.45] and studied in situ hemodynamics at baseline and increased inotropy. Hearts from mTnI mice exhibited increased contractility, as shown by a 30% greater +dP/dt and 18% greater -dP/dt than FVB hearts, and had a negligible response to isoproterenol compared with FVB mice, in which +dP/dt increased by 33% and -dP/dt increased by 26%. Treatment with phenylephrine and propranolol gave a similar result; FVB mouse hearts demonstrated a 20% increase in developed pressure, whereas mTnI mice showed no response. Back phosphorylation of TnI from mTnI hearts demonstrated that the mutation of the PKC sites was associated with an enhanced PKA-dependent phosphorylation independent of a change in basal cAMP levels. Our results demonstrate the important role that PKC-dependent phosphorylation of TnI has on the modulation of cardiac function under basal as well as augmented states and indicate interdependence of the phosphorylation sites of TnI in hearts beating in situ. troponin; protein kinase C; contractility; mouse

Protein kinase C (PKC)-mediated phosphorylation of cardiac myofilament (MF) proteins has been shown to depress the actomyosin interaction and may be important during heart failure. Biochemical studies indicate that phosphorylation of Set (43) and [Ser.sup.45] of cardiac troponin I (cTnI) plays a substantial role in the PKC-mediated depression. We studied intact and detergent-extracted papillary muscles from nontransgenic (NTG) and transgenic (TG) mouse hearts that express a mutant cTnI (Ser43Ala, Ser45Ala) that lacks specific PKC-dependent phosphorylation sites. Treatment of NTG papillary muscles with phenylephrine (PE) resulted in a transient increase and a subsequent 62% reduction in peak twitch force. TG muscles showed no transient increase and only a 45% reduction in force. There was a similar difference in maximum tension between NTG and TG fiber bundles that had been treated with a phorbol ester and had received subsequent detergent extraction. Although levels of cTnI phosphorylation correlated with these differences, the TG fibers also demonstrated a decrease in phosphorylation of cardiac troponin T. The PKC-specific inhibitor chelerythrine inhibited these responses. Our data provide evidence that specific PKC-mediated phosphorylation of [Ser.sup.43] and [Ser.sup.45] of cTnI plays an important role in regulating force development in the intact myocardium. protein kinase C; troponin I

We evaluated the direct effect of hyperglycemia on apoptosis of adult rat ventricular myocytes (ARVM) in vitro. Hyperglycemia (16.5 mM) for 24 h increased apoptosis by greater than threefold (48.2 [+ or -] 4.4%, by the TdT-mediated dUTP nick-end labeling method) compared with baseline (14.7 [+ or -] 2.5%). Hyperosmolarity with mannitol (11.0 mM) in the presence of 5.5 mM glucose also increased apoptosis by approximately twofold of baseline. Both glucose and mannitol treatment resulted in the membrane translocation of protein kinase C (PKC)-[delta], and the activation of PKC-[delta] was confirmed by immune complex kinase assay. PKC-[delta]-specific translocation inhibitor peptide ([delta]V1-1) attenuated only apoptosis induced by hyperglycemia but not by mannitol. A PKC-[epsilon]-specific translocation inhibitor peptide ([epsilon]V1-1) affected neither type of apoptosis. Moderate overexpression of PKC-[delta] by adenovirus gene transfer prevented the antiapoptotic effect of [delta]V1-1. Furthermore, [delta]V1-1 attenuated the production of reactive oxygen species (ROS) by glucose. Taken together, our results indicate that increased ROS production regulated by PKC-[delta] is in part responsible for the induction of apoptosis by hyperglycemia and that apoptosis by hyperglycemia is mechanistically different from that by hyperosmolarity. reactive oxygen species; glucose; mannitol; antioxidant; adenovirus

The cardiac L-type calcium current ([I.sub.Ca]) can be modified by activation of protein kinase C (PKC). However, the effect of PKC activation on [I.sub.Ca] is still controversial. Some studies have shown a decrease in current, whereas other studies have reported a biphasic effect (an increase followed by a decrease in current or vice versa). A possible explanation for the conflicting results is that several isoforms of PKC with opposing effects on [I.sub.Ca] were activated simultaneously. Here, we examined the influence of a single PKC isoform (PKC-[beta]II) on L-type calcium channels in isolation from other cardiac isoforms, using a transgenic mouse that conditionally expresses PKC-[beta]II. Ventricular cardiac myocytes were isolated from newborn mice and examined for expression of the transgene using single cell RT-PCR after [I.sub.Ca] recording. Cells expressing PKC-[beta]II showed a twofold increase in nifedipine-sensitive [I.sub.Ca]. The PKC-[beta]II antagonist LY-379196 returned [I.sub.Ca] amplitude to levels found in non-PKC-[beta]II-expressing myocytes. The increase in [I.sub.Ca] was independent of [Ca.sub.v]1.2-subunit mRNA levels as determined by quantitative RT-PCR. Thus these data demonstrate that PKC-[beta] is a potent modulator of cardiac L-type calcium channels and that this specific isoform increases [I.sub.Ca] in neonatal ventricular myocytes. second messengers; signal transduction

Protein kinase C-[zeta] modulates thromboxane [A.sub.2]-mediated apoptosis in adult ventricular myocytes via Akt. Am J Physiol Heart Circ Physiol 282: H320-H327, 2002.--We hypothesized that thromboxane [A.sub.2] (Tx[A.sub.2]) receptor stimulation directly induces apoptosis in adult cardiac myocytes. To investigate this, we exposed cultured adult rat ventricular myocytes (ARVM) to a Tx[A.sub.2] mimetic [1S-[1[alpha],2[alpha](Z),3[beta](1E,3[S.sup.*]), 4[alpha]]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl] -7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP) for 24 h. Stimulation with I-BOP induced apoptosis in a dose-dependent manner and was completely prevented by a Tx[A.sub.2] receptor antagonist, SQ-29548. We further investigated the role of protein kinase C (PKC) in this process. Tx[A.sub.2] stimulation resulted in membrane translocation of PKC-[zeta] but not PKC-[alpha], -[beta]II, -[delta], and -[member of] at 3 min and 1 h. The activation of PKC-[zeta] by I-BOP was confirmed using an immune complex kinase assay. Treatment of ARVM with a cell-permeable PKC-[zeta] pseudosubstrate peptide ([zeta]-PS) significantly attenuated apoptosis by I-BOP. In addition, I-BOP treatment decreased baseline Akt activity and its decrease was reversed by treatment with [zeta]-PS. The inhibition of phosphatidylinositol 3-kinase upstream of Akt by wortmannin or LY-294002 abolished the antiapoptotic effect of [zeta]-PS. Therefore, our results suggest that the activation of PKC-[zeta] modulates Tx[A.sub.2] receptor-mediated apoptosis at least, in part, through Akt activity in adult cardiac myocytes. atypical protein kinase C; eicosanoids; Akt/protein kinase B; mitogen activated protein kinases

The expression of protein kinase C (PKC) isoforms in fetal, neonatal and adult rat ventricular myocardial tissues was characterized by Northern blot analysis to determine the mechanisms that regulate PKC isoform expression. Northern blot analysis of rat ventricular myocardial tissues indicated the presence of coordinate postnatal declines in PKC-alpha and PKC-delta isoforms during myocardial development. The PKC-lambda transcript was also abundant in neonatal rat hearts which also contained the highest levels of PKC immunoreactivity.

The plasticity of creatine kinase (CrK) isoenzyme system was analyzed in isolated perfused rat hearts after chronic swimming. In vitro enzymatic analysis of trained isolated perfused rat hearts indicated the presence of more rapid recovery of function during reoxygenation and higher CrK activity compared to control rats. Furthermore, damage due to metabolic stress was reduced in trained rat hearts compared to sedentary rats indicating the role of physical training the reducing hypoxia-induced tissue damage.

High coronary perfusion pressure (CPP) in isolated isovolumic heart preparation increases the steady state c-fos mRNA synthesis and translocation and activation of protein kinase C(sub -epsilon). Increased left ventricular end-diastolic pressure in the absence of increased CPP is not associated with an increase in c-fos mRNA expression. Endogenous renin-angiotensin system (RAS) is involved in the induction of c-fos gene expression in the adult perfused heart. This shows that myocardial stretch and RAS are involved in development of cardiac hypertrophy.

Experimental studies of the effect of ventricular pacing on cardiac atrophy and protein synthesis in rat heart show that ventricular pacing reduces cardiac atrophy, protein degradation and hence the shift in the alpha-myosin heavy chain. The decrease in cardiac mass due to ventricular pacing occurs only till the onset of atrophy, after which pacing has no effect on the protein synthesis rate and isomyosin shift. The changes in the cardiac mass and protein synthesis levels in the paced heart 2 weeks after surgery differs from those in the mass and synthesis levels in the hearts paced 1 week of surgery.

Use of C57/Bl6 adult female mice to characterize the cardiac responses to chronic physical exercise such as swimming reveals that the chronic exercise induces cardiac hypertrophy in the mice without any increase in myofibrillar adenosinetriphosphatase function, cardiac tissue norepinephrine level or cardiac myosin ATPase activity. Swimming, however, elevates the heart weight by 10% and the heart weight-to-body weight ratio by 16%, while the heart rate response is reduced by 20%. The effects of chronic conditioning are suitable for murine models that facilitate the molecular analysis of in vivo adaptive cardiac hypertrophy.