Abstract-It is becoming clear that upregulated protein kinase C (PKC) signaling plays a role in reduced ventricular myofilament contractility observed in congestive heart failure. However, data are scant regarding which PKC isozymes are involved. There is evidence that PKC-␣ may be of particular importance. Here, we examined PKC-␣ quantity, activity, and signaling to myofilaments in chronically remodeled myocytes obtained from rats in either early heart failure or end-stage congestive heart failure. Immunoblotting revealed that PKC-␣ expression and activation was unaltered in early heart failure but increased in end-stage congestive heart failure. Left ventricular myocytes were isolated by mechanical homogenization, Triton-skinned, and attached to micropipettes that projected from a force transducer and motor. Myofilament function was characterized by an active force- [Ca 2ϩ ] relation to obtain Ca 2ϩ -saturated maximal force (F max ) and myofilament Ca 2ϩ sensitivity (indexed by EC 50 ) before and after incubation with PKC-␣, protein phosphatase type 1 (PP1), or PP2a. PKC-␣ treatment induced a 30% decline in F max and 55% increase in the EC 50 in control cells but had no impact on myofilament function in failing cells. PP1-mediated dephosphorylation increased F max (15%) and decreased EC 50 (Ϸ20%) in failing myofilaments but had no effect in control cells. PP2a-dependent dephosphorylation had no effect on myofilament function in either group. Lastly, PP1 dephosphorylation restored myofilament function in control cells hyperphosphorylated with PKC-␣. Collectively, our results suggest that in end-stage congestive heart failure, the myofilament proteins exist in a hyperphosphorylated state attributable, in part, to increased activity and signaling of PKC-␣. Key Words: heart failure Ⅲ protein kinase C-␣ Ⅲ myofilament proteins Ⅲ protein phosphatase type 1 Ⅲ phosphorylation I t has been predicted that the global incidence and prevalence of the clinical syndrome of congestive heart failure (CHF) will continue to rise. 1 The "road" to CHF usually begins with some inciting event (eg, myocardial infarction), which imposes a heightened mechanical strain on the myocardium. Ventricular dysfunction ensues resulting in a decline in cardiac output. In turn, key regulatory neurohormonal signals are recruited, which, in the acute phase, maintain cardiac output and "mask" the underlying ventricular contractile deficit. However, prolonged exposure of the heart to these signals coupled with the prevailing mechanical overload proves deleterious resulting in contractile dysfunction, myocyte hypertrophy, and death, heralding a downward spiral wherein ventricular dysfunction becomes manifest and the clinical features of CHF overt. Not surprisingly, considerable attention is now being focused on unraveling the molecular and cellular complexities that conspire to promote contractile dysfunction of the failing cardiac myocyte, with the underlying aim being identification of novel molecules that may be potential foci for therapeutic inte...

Cysteine-rich protein 3 or muscle LIM protein (MLP) is thought to be a mechanosensor in cardiac myocytes. Therefore, the subcellular location of MLP may have functional implications in health and disease. Our hypothesis is that MLP becomes mislocalized after prolonged overload, resulting in impaired mechanosensing in cardiac myocytes. Using the techniques of biochemical subcellular fractionation and immunocytochemistry, we found MLP exhibits oligomerization in the membrane and cytoskeleton of cultured cardiac rat neonatal myocytes. Nuclear MLP was always monomeric. MLP translocated to the nucleolus in response to 10% cyclic stretch at 1 Hz for 48 h. This was associated with a threefold increase in S6 ribosomal protein (P Ͻ 0.01; n ϭ 3 cultures). Adenoviral overexpression of MLP also resulted in a twofold increase in S6 protein, suggesting that MLP can activate ribosomal protein synthesis in the nucleolus. In ventricles from aortic-banded and myocardially infarcted rat hearts, nuclear MLP increased by twofold (P Ͻ 0.01; n ϭ 7) along with a significant decrease in the nonnuclear oligomeric fraction. The ratio of nuclear to nonnuclear MLP increased threefold in both groups (P Ͻ 0.01; n ϭ 7). In failing human hearts, there was almost a complete loss of oligomeric MLP. Using a flag-tagged adenoviral MLP, we demonstrate that the COOH terminus is required for oligomerization and that this is a precursor to stretch sensing and subsequent nuclear translocation. Therefore, reduced oligomeric MLP in the costamere and cytoskeleton may contribute to impaired mechanosensing in heart failure.hypertrophy; mechanosensing; cytoskeleton; nucleocytoplasmic shuttling PROLONGED HEMODYNAMIC OVERLOAD results in cardiac hypertrophy with detrimental changes in myocardial gene expression and morphology (7,8,19,20,24,32) that predict morbidity and mortality (9, 28). Stretch sensors, necessary for myocyte enlargement, are thought to become impaired as a result of prolonged overload (11). However, the mechanisms by which chronic overload leads to decompensation and failure are poorly understood.Many proteins shuttle from subcellular compartments to the nucleus and may act as molecular sensors (4). In myocytes, some of these nucleocytoplasmic proteins are located in costameric focal adhesions and the cytoskeleton where mechanical forces are transmitted. One such protein, muscle LIM protein (MLP) or cysteine-rich protein 3, contains two zinc finger LIM domains. MLP is known to interact with many proteins of the cytoskeleton, including ␣-actinin and the titin-binding protein telethonin (34). MLP is thought to be part of a mechanosensing mechanism in myocytes, and this is likely to occur through two main mechanisms. First, the LIM domains enable the protein to interact with a variety of other cellular proteins, many of which are associated with known signaling pathways. The potential role of MLP as a sensor of mechanical activity through its interactions with other proteins has been thoroughly reviewed recently (18). Second, the protein contains...

Overall, adherence to current nutrient guidelines (as indexed by the DMI) are associated with lower total CVD risk, and additional dietary factors (as indexed by the AHEI) were associated with a lower risk of CVD and HF.

(LVH) or congestive heart failure (CHF). To address this issue, we studied pressure overload-induced LV hypertrophy (POLVH) and myocardial infarction-elicited congestive heart failure (MICHF) in rats. LV myocytes were isolated from control, POLVH, and MICHF hearts by mechanical homogenization, skinned with Triton, and attached to micropipettes that projected from a sensitive force transducer and high-speed motor. sensitivity toward levels observed in control cells. In contrast, integration of cTn purified from failing ventricles into control myocytes increased EC50 to levels observed in failing myocytes. The Fmax parameter was not markedly affected by troponin exchange. cTnI phosphorylation was increased in both POLVH and MICHF left ventricles. We conclude that depressed myofilament Ca 2ϩ sensitivity in experimental LVH and CHF is due, in part, to a decreased functional role of cTn that likely involves augmented phosphorylation of cTnI. left ventricle; troponin; phosphorylation; cardiac disease CONGESTIVE HEART FAILURE (CHF) is characterized by reduced ventricular pump function, which is due, in part, to cardiac myocyte dysfunction. It has been widely reported that Ca 2ϩ homeostasis is impaired in CHF (14). However, whether depressed myofilament function contributes to reduced ventricular myocyte contractility in CHF is less clear (5). Studies probing myofilament activation in failing human myocardium must be interpreted with caution because tissue quality, pharmacological treatment, and brain death of donors may confound experimental findings (15,30,44). For these reasons, investigators have employed animal models that allow for the study of myofilament function under more carefully controlled circumstances. For instance, studies in the pacing-induced canine model of CHF indicate that the myofilaments generate more force for a given level of activator Ca 2ϩ (increased Ca 2ϩ sensitivity) compared with controls (45). Examination of myofilament activity in the spontaneously hypertensive heart failure prone (SHHF) rat demonstrates that myofilament function is either augmented or unchanged depending on when studies are performed during the disease progression (32). In contrast, Pérez and coworkers (31) found reduced myofilament function in right ventricular (RV) trabeculae of the SHHF rat. Similarly, de Tombe et al. (7) have also shown reduced myofilament function in RV trabeculae obtained from rats with large left ventricular (LV) infarcts and in skinned RV myocytes isolated from rats with chronic RV hypertrophy induced by pulmonary artery banding (9). However, the impact of experimental LV hypertrophy (LVH) or CHF on myofilament function in the more clinically relevant left ventricle has not been carefully studied. The molecular basis for altered myofilament function in LVH and CHF likely involves changes in thick and thin filament proteins. It has been reported that protein kinase C (PKC) is upregulated in cardiac disease (6,13,43). In addition, recent work from our group indicates that PKC-mediated phosphoryla...

Background Whether fish or the fatty acids they contain are independently associated with risk for incident heart failure (HF) among postmenopausal women is unclear. Methods and Results The baseline Women’s Health Initiative Observational Study (WHI-OS) cohort consisted of 93,676 women aged 50–79 of diverse ethnicity and background of which 84,493 were eligible for analyses. Intakes of baked/broiled fish, fried fish and omega-3 fatty acid (eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA), α-linolenic acid (ALA)), and trans fatty acid (TFA) were determined from the WHI food frequency questionnaire. Baked/broiled fish consumption was divided into 5 frequency categories: <1/mo (referent), 1–3/mo, 1–2/wk, 3–4/wk, ≥5/wk. Fried fish intake was grouped into 3 frequency categories: <1/mo (referent), 2) 1–3/mo, and 3) ≥1/wk. Associations between fish or fatty acid intake and incident HF were determined using Cox models adjusting for HF risk factors and dietary factors. Baked/broiled fish consumption (≥5 servings/wk at baseline) was associated with a hazard ratio (HR) of 0.70 (95% CI: 0.51, 0.95) for incident HF. In contrast, fried fish consumption (≥1 serving/wk at baseline) was associated with a HR of 1.48 (95% CI: 1.19, 1.84) for incident HF. No significant associations were found between EPA+DHA, ALA, or TFA intake and incident HF. Conclusions Increased baked/broiled fish intake may lower HF risk, while increased fried fish intake may increase HF risk in postmenopausal women.

The PVs and PLA demonstrate unique activation and repolarization characteristics in response to autonomic manipulation. The heterogeneity of vagal responses correlates with the pattern of IKAch distribution in the LA. The peculiar autonomic characteristics of the PVs and PLA might create substrate for re-entry and AF.

The importance of magnesium intake in relation to the metabolic syndrome has been increasingly recognized. Magnesium is an essential mineral, critical for a number of metabolic functions in the human body. The major dietary sources of magnesium intake include whole grains, legumes, nuts, and green leafy vegetables. Animal studies indicate a pivotal role of magnesium in glucose homeostasis and insulin secretion and action. Experimental and clinical studies suggest that magnesium intake may be inversely related to the risk of hypertension and type 2 diabetes mellitus, and may decrease blood triglyceride and increase high-density lipoprotein cholesterol levels. The purpose of this brief review is to summarize the epidemiologic data relating magnesium to the metabolic syndrome and to discuss the potential mechanisms.

This study was conducted to identify molecular mechanisms which explain interventricular differences in myofilament function in experimental congestive heart failure (CHF). CHF was induced in rats by chronic aortic banding or myocardial infarction for 32–36 weeks. Right and left ventricular (RV, LV) myocytes were mechanically isolated, triton-skinned, and attached to a force transducer and motor arm. Myofilament force–[Ca2+] relations assessed maximal Ca2+-saturated force (Fmax) and the [Ca2+] at 50% of Fmax (EC50). Myofilament protein phosphorylation was determined via ProQ diamond phospho-staining. Protein kinase C (PKC)-α expression/activation and site-specific phosphorylation of cardiac troponin I (cTnI) and cardiac troponin T (cTnT) were measured via immunoblotting. Relative to controls, failing RV myocytes displayed a ~45% decrease in Fmax with no change in EC50, whereas failing LV myocytes displayed a ~45% decrease in Fmax and ~50% increase in EC50. Failing LV myofilaments were less Ca2+-sensitive (37% increase in EC50) than failing RV myofilaments. Expression and activation of PKC-α was increased twofold in failing RV myocardium and relative to the RV, PKC-α was twofold higher in the failing LV, while PKC-β expression was unchanged by CHF. PKC-α-dependent phosphorylation and PP1-mediated dephosphorylation of failing RV myofilaments increased EC50 and increased Fmax, respectively. Phosphorylation of cTnI and cTnT was greater in failing LV myofilaments than in failing RV myofilaments. RV myofilament function is depressed in experimental CHF in association with increased PKC-α signaling and myofilament protein phosphorylation. Furthermore, myofilament dysfunction is greater in the LV compared to the RV due in part to increased PKC-α activation and phosphorylation of cTnI and cTnT.