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The existence of a cardiac renin-angiotensin system, independent of the circulating renin-angiotensin system, is still controversial. We compared the tissue levels of reninangiotensin system components in the heart with the levels in blood plasma in healthy pigs and 30 hours after nephrectomy. Angiotensin I (Ang I)-generating activity of cardiac tissue was identified as renin by its inhibition with a specific active site-directed renin inhibitor. We took precautions to prevent the ex vivo generation and breakdown of cardiac angiotensins and made appropriate corrections for any losses of intact Ang I and II during extraction and assay. Tissue levels of renin (n=ll) and Ang I (n=7) and II (n=7) in the left and right atria were higher than in the corresponding ventricles (P< .05). Cardiac renin and Ang I levels (expressed per gram wet weight) were similar to the plasma levels, and Ang II in cardiac tissue was higher than in plasma (P<.05). The presence of these renin-angiotensin system components in cardiac tissue therefore cannot be accounted for by trapped plasma or simple diffusion from plasma into the interstitial fluid. Angiotensinogen levels (n=ll) in cardiac tissue were 10% to 25% of the A ngiotensin I (Ang I) is produced in the circulating / \ blood by the action of renin from the kidney on -Z A . angiotensinogen produced by the liver. Ang I is converted to Ang II, a potent vasoconstrictor, by angiotensin-converting enzyme (ACE) located on the luminal surface of the vascular endothelium. It is now well established that Ang I and II are not only produced in the blood compartment but also locally in tissues. Recent evidence suggests that complete local reninangiotensin systems (RAS) are present in a number of organs, for instance, kidney, adrenal gland, and ovary. 13In such local RAS, the production of Ang I and II is thought to depend on in situ synthesized renin rather than plasma-derived renin.A local cardiac RAS has also been postulated. 4 ' 5 However, direct evidence for Ang I and II production in the heart by in situ synthesized renin is still lacking. Renin mRNA levels in the heart are usually low and can be detected only by polymerase chain reaction. 68 Early studies showed Ang I-generating activity in left ventric-

Background-Mutations in the MYBPC3 gene, encoding cardiac myosin-binding protein C (cMyBP-C), are a frequent cause of familial hypertrophic cardiomyopathy. In the present study, we investigated whether protein composition and function of the sarcomere are altered in a homogeneous familial hypertrophic cardiomyopathy patient group with frameshift mutations in MYBPC3 (MYBPC3 mut ). Methods and Results-Comparisons were made between cardiac samples from MYBPC3 mutant carriers (c.2373dupG, nϭ7; c.2864_2865delCT, nϭ4) and nonfailing donors (nϭ13). Western blots with the use of antibodies directed against cMyBP-C did not reveal truncated cMyBP-C in MYBPC3 mut . Protein expression of cMyBP-C was significantly reduced in MYBPC3 mut by 33Ϯ5%. Cardiac MyBP-C phosphorylation in MYBPC3 mut samples was similar to the values in donor samples, whereas the phosphorylation status of cardiac troponin I was reduced by 84Ϯ5%, indicating divergent phosphorylation of the 2 main contractile target proteins of the ␤-adrenergic pathway. Force measurements in mechanically isolated Triton-permeabilized cardiomyocytes demonstrated a decrease in maximal force per crosssectional area of the myocytes in MYBPC3 mut (20.2Ϯ2.7 kN/m 2 ) compared with donor (34.5Ϯ1.1 kN/m 2 ). Moreover, Ca 2ϩ sensitivity was higher in MYBPC3 mut (pCa 50 ϭ5.62Ϯ0.04) than in donor (pCa 50 ϭ5.54Ϯ0.02), consistent with reduced cardiac troponin I phosphorylation. Treatment with exogenous protein kinase A, to mimic ␤-adrenergic stimulation, did not correct reduced maximal force but abolished the initial difference in Ca 2ϩ sensitivity between MYBPC3 mut (pCa 50 ϭ5.46Ϯ0.03) and donor (pCa 50 ϭ5.48Ϯ0.02). Conclusions-Frameshift MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins, and reduced maximal force-generating capacity of cardiomyocytes. The enhanced Ca 2ϩ sensitivity in MYBPC3 mut is due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction.

Abstract-Tissue accumulation of circulating prorenin results in angiotensin generation, but could also, through binding to the recently cloned (pro)renin receptor, lead to angiotensin-independent effects, like p42/p44 mitogen-activated protein kinase (MAPK) activation and plasminogen-activator inhibitor (PAI)-1 release. Here we investigated whether prorenin exerts angiotensin-independent effects in neonatal rat cardiomyocytes. Polyclonal antibodies detected the (pro)renin receptor in these cells. Prorenin affected neither p42/p44 MAPK nor PAI-1. PAI-1 release did occur during coincubation with angiotensinogen, suggesting that this effect is angiotensin mediated. Prorenin concentrationdependently activated p38 MAPK and simultaneously phosphorylated HSP27. The latter phosphorylation was blocked by the p38 MAPK inhibitor SB203580. Rat microarray gene (nϭ4800) transcription profiling of myocytes stimulated with prorenin detected 260 regulated genes (PϽ0.001 versus control), among which genes downstream of p38 MAPK and HSP27 involved in actin filament dynamics and (cis-)regulated genes confined in blood pressure and diabetes QTL regions, like Syntaxin-7, were overrepresented. Quantitative real-time RT-PCR of 7 selected genes (Opg, Timp1, Best5, Hsp27, Col3a1, and Hk2) revealed temporal regulation, with peak levels occurring after 4 hours of prorenin exposure. This regulation was not altered in the presence of the renin inhibitor aliskiren or the angiotensin II type 1 receptor antagonist eprosartan. Finally, pilot 2D proteomic differential display experiments revealed actin cytoskeleton changes in cardiomyocytes after 48 hours of prorenin stimulation. In conclusion, prorenin exerts angiotensinindependent effects in cardiomyocytes. Prorenin-induced stimulation of the p38 MAPK/HSP27 pathway, resulting in alterations in actin filament dynamics, may underlie the severe cardiac hypertrophy that has been described previously in rats with hepatic prorenin overexpression. (Hypertension. 2006;48:564-571.)

Myocardial infarction (MI) initiates cardiac remodeling, depresses pump function, and predisposes to heart failure. This study was designed to identify early alterations in Ca2+ handling and myofilament proteins, which may contribute to contractile dysfunction and reduced beta-adrenergic responsiveness in postinfarct remodeled myocardium. Protein composition and contractile function of skinned cardiomyocytes were studied in remote, noninfarcted left ventricular (LV) subendocardium from pigs 3 weeks after MI caused by permanent left circumflex artery (LCx) ligation and in sham-operated pigs. LCx ligation induced a 19% increase in LV weight, a 69% increase in LV end-diastolic area, and a decrease in ejection fraction from 54+/-5% to 35+/-4% (all P<0.05), whereas cardiac responsiveness to exercise-induced increases in circulating noradrenaline levels was blunted. Endogenous protein kinase A (PKA) was significantly reduced in remote myocardium of MI animals, and a negative correlation (R=0.62; P<0.05) was found between cAMP levels and LV weight-to-body weight ratio. Furthermore, SERCA2a expression was 23% lower after MI compared with sham. Maximal isometric force generated by isolated skinned myocytes was significantly lower after MI than in sham (15.4+/-1.5 versus 19.2+/-0.9 kN/m2; P<0.05), which might be attributable to a small degree of troponin I (TnI) degradation observed in remodeled postinfarct myocardium. An increase in Ca2+ sensitivity of force (pCa50) was observed after MI compared with sham (DeltapCa50=0.17), which was abolished by incubating myocytes with exogenous PKA, indicating that the increased Ca2+ sensitivity resulted from reduced TnI phosphorylation. In conclusion, remodeling of noninfarcted pig myocardium is associated with decreased SERCA2a and myofilament function, which may contribute to depressed LV function. The full text of this article is available online at http://circres.ahajournals.org.

Abstract-The extent and mechanism of the cardiac benefit of early exercise training following myocardial infarction (MI) is incompletely understood, but may involve blunting of abnormalities in Ca 2ϩ -handling and myofilament function. Consequently, we investigated the effects of 8-weeks of voluntary exercise, started early after a large MI, on left ventricular (LV) remodeling and dysfunction in the mouse. Exercise had no effect on survival, MI size or LV dimensions, but improved LV fractional shortening from 8Ϯ1 to 12Ϯ1%, and LVdP/dt P30 from 5295Ϯ207 to 5794Ϯ207 mm Hg/s (both PϽ0.05), and reduced pulmonary congestion. These global effects of exercise were associated with normalization of the MI-induced increase in myofilament Ca 2ϩ -sensitivity (⌬pCa 50 ϭ0.037). This effect of exercise was PKA-mediated and likely because of improved ␤ 1 -adrenergic signaling, as suggested by the increased ␤ 1 -adrenoceptor protein (48%) and cAMP levels (36%; all PϽ0.05). Exercise prevented the MI-induced decreased maximum force generating capacity of skinned cardiomyocytes (F max increased from 14.3Ϯ0.7 to 18.3Ϯ0.8 kN/m 2 PϽ0.05), which was associated with enhanced shortening of unloaded intact cardiomyocytes (from 4.1Ϯ0.3 to 7.0Ϯ0.6%; PϽ0.05). Furthermore, exercise reduced diastolic Ca 2ϩ -concentrations (by ϳ30%, PϽ0.05) despite the unchanged SERCA2a and PLB expression and PLB phosphorylation status. Importantly, exercise had no effect on Ca 2ϩ -transient amplitude, indicating that the improved LV and cardiomyocyte shortening were principally because of improved myofilament function. In conclusion, early exercise in mice after a large MI has no effect on LV remodeling, but attenuates global LV dysfunction. The latter can be explained by the exercise-induced improvement of myofilament function. (Circ Res. 2007;100:1079-1088.)Key Words: cardiac function Ⅲ cardiomyocytes Ⅲ exercise training Ⅲ heart failure L eft ventricular (LV) remodeling after myocardial infarction (MI) is a compensatory mechanism that serves to restore LV pump function. Despite the apparent appropriateness of LV remodeling to maintain cardiac pump function early after MI, remodeling is an independent risk factor for the development of congestive heart failure. 1 The mechanism underlying the progression from LV remodeling to overt heart failure remains incompletely understood, but recent evidence indicates that abnormalities in myofilament function and Ca 2ϩ -handling contribute to the LV dysfunction in the porcine heart, early after MI. 2 In contrast to pathological LV remodeling after MI, LV remodeling produced by regular dynamic exercise is associated with a decreased risk for coronary artery disease and heart failure. 3 Exercise training is associated with an increased myocardial perfusion capacity and with normal or even increased contractile function in the normal heart. 4,5 There is also clinical evidence that exercise after MI has a beneficial effect on disease progression and survival. 6,7 For example, physical conditioning in patients with L...

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the four important S4 arginine residues on the voltage-sensing paddle is still unknown, with some models placing them in an aqueous crevice, and others a lipid environment. To learn more about the intricate role of lipid in the structure and function of potassium channels we have studied deuterium and phosphate ESEEM on spin-labeled, liposome reconstituted KcsA. By scanning the trans-membrane helices of KcsA, we show that deuterium coupling can be used to determine residue depth within a lipid bilayer. In addition, residues that interact with the phosphate head-groups of the lipid can be determined by phosphate coupling, and their precise location modeled.