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(Circulation Research. 2007;101:195.)
© 2007 American Heart Association, Inc.

Integrative Physiology

Augmented Protein Kinase C-–Induced Myofilament Protein Phosphorylation Contributes to Myofilament Dysfunction in Experimental Congestive Heart Failure

Rashad J. Belin, Marius P. Sumandea, Edward J. Allen, Kelly Schoenfelt, Helen Wang, R. John Solaro, Pieter P. de Tombe

From the Department of Physiology and Biophysics, Center for Cardiovascular Research, University of Illinois at Chicago.

Correspondence to Dr Pieter P. de Tombe, PhD, University of Illinois at Chicago, Department of Physiology and Biophysics, 835 S Wolcott (M/C 901), Chicago, IL 60612. E-mail pdetombe{at}uic.edu

	Abstract

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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–[Ca²⁺] relation to obtain Ca²⁺-saturated maximal force (F_max) and myofilament Ca²⁺ sensitivity (indexed by EC₅₀) 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 EC₅₀ in control cells but had no impact on myofilament function in failing cells. PP1-mediated dephosphorylation increased F_max (15%) and decreased EC₅₀ (20%) 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 • protein kinase C- • myofilament proteins • protein phosphatase type 1 • phosphorylation

	Introduction

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It 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 intervention. One promising therapeutic target is the multifunctional protein kinase C (PKC) signaling system. PKC comprises a family of serine/threonine kinases activated by mechanical and neurohormonal signals that are involved in the management of a myriad of cellular processes including: transcription, cell growth, cell death, Ca²⁺-handling, and myofilament function.^2–5

Recent studies have suggested that PKC-mediated, site-specific phosphorylation of cardiac troponin (cTn)I and cTnT depresses myofilament contractility, intrinsic myocyte function, and ventricular pump function.^4–10 Moreover, we and others have observed that, in various animal models of end-stage cardiac failure (eg, myocardial infarction, pressure overload) and in different cardiac muscle preparations (eg, cells and trabeculae) isolated from different regions of the myocardium (right versus left ventricle), myofilament function is severely depressed.^11–17 Furthermore, in a recent report using 2 distinct rat models of ventricular failure, we observed depressed myofilament function secondary to dysfunction of the regulatory cTn complex and increased phosphorylation of cTnI.¹¹ Our central tenet is that prolonged excess mechanical stress/strain on the myocyte activates several PKC isozymes, a process that, in addition to promoting cell growth and death, also triggers functionally important phosphorylations of several myofilament proteins causing depressed myofilament and, consequently, myocyte function. Indeed, several studies have shown that activity and expression of numerous PKC isozymes is upregulated in experimental and human CHF.^18–20

Once activated, there are several distinct PKC isozymes that would affect the cardiac sarcomere to elicit myofilament dysfunction in failing hearts.^7,21,22 Recently, PKC- has emerged as a key player in contractile dysfunction, development of heart failure (HF), and control of myofilament activity. Studies from Braz et al indicate that depletion of myocardial PKC- results in increased myocardial contractility, whereas transgenic overexpression of the molecule leads to marked ventricular dysfunction.²³ These investigators proposed that myocyte dysfunction in the PKC- transgenic mouse is caused by alterations in Ca²⁺ homeostasis.²³ Both cTnI and cTnT are viable substrates for PKC-–dependent phosphorylation,^4,5,24 and studies by Sumandea et al⁵ demonstrate that PKC- depresses myofilament contractility through site-specific phosphorylation of cTnT at the threonine-206 residue. Thus, it is plausible that augmented signaling through PKC- elicits myofilament dysfunction of the failing myocyte through phosphorylation of cTnI and/or cTnT. However, studies examining the functional link between PKC- and myofilament dysfunction in failing ventricles are lacking. Accordingly, we examined PKC- expression, activation, and impact on myofilament function in skinned left ventricular myocytes isolated from rats subjected to chronic (8 to 9 months) pressure overload and myocardial infarction; experimental CHF models in which myofilament function is severely depressed.¹¹ We also examined PKC- expression and activity in rats at an early stage of HF to elucidate the role of PKC- in mediating the transition to end-stage CHF. To assess whether myofilament protein hyperphosphorylation causes the depressed myofilament phenotype in experimental CHF, we determined whether dephosphorylation of failing myofilaments with protein phosphatase type 1 (PP1) or PP2a could rescue myofilament force development and Ca²⁺sensitivity. Here, we report that PKC- expression and activity was unaltered in early HF but was similarly upregulated in 2 distinct rat models of end-stage CHF. In skinned myocytes, PKC-–dependent phosphorylation resulted in marked depression of control myofilament function but had no impact on failing myofilaments. Also, PP1-elicited dephosphorylation increased myofilament function in failing myocytes and control cells hyperphosphorylated with PKC- but had no effect in untreated control cells. Our data indicate that myofilament dysfunction in end-stage CHF arises, in part, from increased myofilament protein phosphorylation through PKC-.

	Materials and Methods

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Animal Models of Ventricular Hypertrophy and CHF
All procedures used were in accordance with the institutional guidelines regarding the care and use of laboratory animals. Ascending aortic banding and myocardial infarction (MI) were performed on 4-week female Sprague–Dawley rats as described previously, with slight modifications.^11,15 Animals in the early HF cohort were followed for a period of 12 weeks,²⁵ whereas animals in the end-stage CHF group were followed for 32 to 36 weeks until they transitioned to end-stage CHF. Unoperated age-matched animals served as controls. Previously, we found no difference between sham operated and age-matched control animals.^11,14

Force–[Ca²⁺] Measurements in Skinned Ventricular Myocytes
Myocytes were isolated from the interventricular septum and left ventricular free wall of CHF, left ventricular hypertrophy (LVH), and age-matched control hearts by mechanical homogenization and chemically permeabilized (skinned) with 0.3% Triton X-100.^11,14 We observed no difference in myofilament function between septal and left ventricular myocytes.¹¹ Details of the solutions for cell isolation and experimentation have been previously described in detail.^11,14,26 Cells were stored on ice and used within 20 hours of isolation. Sarcomere length was set to 2.10 µm by video micrometry.¹¹

PKC-, PP1, and PP2a Expression
Approximately 30 to 40 mg of left ventricular early HF, LVH, CHF, and age-matched control myocardium was homogenized in homogenization buffer (20 mmol/L HEPES, 150 mmol/L NaCl, 15% [vol/vol] glycerol, 5 mmol/L MgCl₂, 1 mmol/L EGTA, 1 mmol/L EDTA, 1 mmol/L Na₃VO₄, 100 mmol/L NaF, 10 mmol/L Na-pyrophosphate, 1% [vol/vol] Triton X-100, 1% [vol/vol] Na-deoxycholate, 1 mmol/L dithiothreitol, 0.1% [vol/vol], 1 mmol/L 4-[2-aminoethyl]benzenesulfonyl fluoride, 50 µg/mL aprotinin, 5 mmol/L pepstatin A, and 50 µg/mL leupeptin) on ice. The homogenate was centrifuged at 4°C and 100 000g for 1 hour. The protein concentration of the supernatant was determined and the samples were stored at –80°C. A total of 150 µg of protein was loaded onto 10% SDS-PAGE gels. The electrophoresed proteins were transferred to poly(vinylidene difluoride) membranes. Membranes were incubated with primary antibodies against PKC-, phospho-specific PKC-, PKC-ß, PKC-, PKC-, PP1, and PP2a (Upstate Biotechnology). Secondary anti-mouse and anti-rabbit IgG peroxidase conjugates (Sigma-Aldrich) were used. The relative abundance of single proteins was detected using enhanced chemiluminescence (Amersham Biosciences). Suitable films were scanned and PKC-, phospho-specific PKC-, PKC-ß, PKC-, PKC-, PP1, PP2a, and actin band density was quantified using commercially available software (Image J; NIH). Scanning units were normalized to actin units.

Expression and Purification of Recombinant PKC-
Expression and purification of recombinant human PKC- was executed as described previously in detail.⁵

PKC-–Mediated Phosphorylation and PP1- and PP2a-Mediated Dephosphorylation of Myofilament Proteins in Ventricular Myocytes
To assess the impact of PKC- mediated phosphorylation on myofilament function an attached cell was washed with PKC- buffer less the enzyme for 2 minutes. The cell was then incubated in PKC- buffer (1 mmol/L NaF, 1 mmol/L Na₃VO₄, 0.5 mmol/L MgCl₂, 100 mmol/L leupeptin, 100 mmol/L pepstatin, 150 mmol/L PMSF·EtOH, 1 mmol/L dithiothreitol, 0.8 µmol/L Ca²⁺, 20 µmol/L diacylglycerol [DAG], 0.3 mmol/L phosphatidylserine [PS], and 0.1 µg/mL recombinant PKC-) at 22°C to 25°C for 60 minutes. For PP1- and PP2a-induced dephosphorylation, myocytes were incubated in relaxing solution containing the catalytic subunit of PP1 (0.15 U/mL; Upstate Biotechnology) or PP2a (0.15 U/mL; Upstate Biotechnology) along with 1 mmol/L dithiothreitol at 22°C to 25°C for 60 minutes. Following the incubation, the attached cell was washed and exposed to 4 submaximal Ca²⁺ concentrations.

Data and Statistical Analyses
Cell data were analyzed as described earlier.^11,14,26 Data are expressed as means±SEM. Statistical differences in PKC-, PP1, and PP2a expression between early HF, end-stage CHF, and age-matched control ventricles were determined using unpaired Student’s t test. The impact of lipids, PKC-, PP1, and PP2a on myofilament function was determined by a paired Student’s t test with a P<0.05 considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
PKC- Isozyme Expression in Experimental Early HF and End-Stage CHF
Left ventricles obtained from early HF, end-stage CHF, and age-matched control hearts were analyzed by immunoblotting to determine relative expression levels of PKC-. Because of the high amount of detergent used in the homogenization buffer, it is unlikely that much of the PKC remained in the particulate (myofilament) fraction, and thus the amount of PKC protein in the detergent extracted fraction represents most of the PKC found inside the ventricular myocyte. At an early stage of HF, characterized by preserved myofilament function,²⁵ we found no changes in PKC- expression or activation (Figure 1A and 1B). In contrast, PKC- expression was increased 4-fold in CHF relative to age-matched control ventricles (Figure 1C). Similarly, LVH resulted in 3-fold upregulation in PKC- expression. Furthermore, activation of PKC-, indexed by autophosphorylated PKC-, was increased 4-fold in both LVH and CHF ventricular myocardium (Figure 1D). We also examined expression of PKC-ß, PKC-, and PKC- and found that, relative to control, there were no increases in expression of these isozymes in end-stage experimental CHF (data not shown). Collectively, these data suggest that PKC- is involved in the transition to end-stage CHF and that regardless of the etiology, there is a similar upregulation of PKC- expression and activity in the rat ventricle.

View larger version (37K): [in this window] [in a new window]   Figure 1. A, PKC- isozyme protein expression. The inset shows representative Western blots of PKC- and actin protein expression in control (CON) (n=8) and early HF (n=8) ventricular samples. B, Western blot showing phosphorylated PKC- expression in the same ventricular samples as in A. C, The inset shows representative Western blots of PKC- and actin protein expression in CHF (n=10), LVH (n=10), and age-matched control (n=10; n=10) ventricular samples. D, Western blot showing phosphorylated PKC- expression in the same ventricular samples as in C. *Significant vs age-matched control for each respective experimental group (P<0.05).

PP1 and PP2a Expression in Experimental Early HF and End-Stage CHF
Figure 2A through 2D illustrates that expression of PP1 and PP2a in early HF and end-stage CHF was unaltered relative to age-matched controls. These data suggest that altered expression of PP1 and PP2a is not involved in early HF or the transition to end-stage CHF in the rat ventricle.

View larger version (37K): [in this window] [in a new window]   Figure 2. A, PP1 protein expression. The inset shows representative Western blots of PP1 and actin protein expression in control (CON) (n=8) and early HF (n=8) ventricular samples. B, Western blot showing PP2a expression in the same ventricular samples as in A. C, The inset shows representative Western blots of PP1 and actin protein expression in CHF (n=10), LVH (n=10), and age-matched control (n=10) ventricular samples. D, Western blot showing PP2a expression in the same ventricular samples as in C.

Effect of PKC-–Mediated Phosphorylation on Myofilament Function
All myocyte experiments were performed on the stage of an inverted microscope (Figure 3).¹¹

View larger version (147K): [in this window] [in a new window]   Figure 3. A, Photomicrograph of an attached ventricular myocyte in relaxing solution at a sarcomere length of 2.10 µm. B, Side view illustrating direct measurement of myocyte height in relaxing solution at a sarcomere length of 2.10 µm.

Figure 4 illustrates the effect of DAG and PS on myofilament function in control cells. Treatment of cardiac cells with the lipids had no appreciable effect on Ca²⁺-saturated maximal force (F_max) and the myofilament Ca²⁺-sensitivity index (EC₅₀) (P>0.05). The effect of PKC- mediated phosphorylation on myofilament function of control cells is delineated in Figure 5A through 5C. PKC-–dependent phosphorylation resulted in a 30% reduction in the F_max parameter and 50% increase in the EC₅₀ (decreased myofilament Ca²⁺ sensitivity). In contrast, the F_max and EC₅₀ in failing cells were not significantly affected by PKC- treatment (Figure 5D through 5F). Furthermore, there was no statistical difference between F_max and EC₅₀ in control cells treated with PKC- and untreated CHF cells (P>0.05). Taken together, these results suggest that increased PKC- mediated myofilament phosphorylation contributes to myofilament dysfunction in end-stage experimental CHF.

View larger version (15K): [in this window] [in a new window]   Figure 4. A, Average force–[Ca2+] relations for control (CON) myocytes (n=5) before (CON-DAG/PS) and after (CON+DAG/PS) incubation with DAG and PS in the PKC myocyte incubation buffer. B and C, Bar graphs of averaged curve fit parameters (Fmax and EC50) from control cells before and after treatment with phospholipids.

View larger version (28K): [in this window] [in a new window]   Figure 5. A, Average force–[Ca2+] relations for control (CON) myocytes (n=10) before (CON-PKC) and after (CON+PKC) incubation with recombinant PKC-. B and C, Histograms of averaged curve fit parameters (Fmax and EC50) from control cells before and after treatment with bacterially expressed PKC-. D, Average force–[Ca2+] relations for CHF myocytes (n=7) before (CHF-PKC) and after (CHF+PKC) incubation with recombinant PKC-. E and F, Histograms of averaged curve fit parameters (Fmax and EC50) from CHF cells before and after treatment with bacterially expressed PKC-.*Significantly different vs CON-PKC (P<0.05).

Impact of PP1- and PP2a-Mediated Dephosphorylation on Myofilament Function
To confirm that augmented phosphorylation contributes to the depressed myofilament phenotype in experimental CHF, we determined the impact of PP1- and PP2a-dependent dephosphorylation on myofilament function in skinned myocytes isolated from control and failing left ventricles (Figures 6 and 7). Incubation of control myofilaments with the catalytic subunit of PP1 had no effect on F_max or EC₅₀ (Figure 6A through 6C). In contrast, PP1-mediated dephosphorylation of failing myofilament proteins resulted in a significant 15% increase in the F_max and 20% decrease in the EC₅₀ parameter (increased myofilament Ca²⁺ sensitivity; Figure 6D through 6F). PP2a-dependent dephosphorylation had no effect on F_max or EC₅₀ in control or CHF cells (Figure 7). Finally, to confirm that increased PKC-–dependent phosphorylation reduces myofilament function, we treated nonfailing cells with PKC- followed by PP1. PKC- treatment resulted in a marked depression of myofilament function (decreased F_max by 30%; increased EC₅₀ by 35%). Importantly, PP1-mediated dephosphorylation normalized myofilament activity and resulted in a 25% increase in F_max and 30% decrease in the EC₅₀ (Figure 8). Overall, our findings strongly suggest that increased PKC-–elicited phosphorylation is partially responsible for myofilament dysfunction in end-stage experimental CHF and that this dysfunctional phenotype can be partially reversed by dephosphorylation with PP1.

View larger version (28K): [in this window] [in a new window]   Figure 6. A, Average force–[Ca2+] relations for control (CON) myocytes (n=9) before (CON-PP1) and after (CON+PP1) incubation with the catalytic subunit of protein PP1 (0.15 U/mL). B and C, Bar graphs of averaged curve fit parameters (Fmax and EC50) from control cells before and after treatment with PP1. D, Average force–[Ca2+] relations for CHF myocytes (n=8) before (CHF-PP1) and after (CHF+PP1) incubation with the catalytic subunit of protein PP1 (0.15 U/mL). E and F, Bar graphs of averaged curve fit parameters (Fmax and EC50) from CHF cells before and after treatment with PP1. *Significant vs CHF (P<0.05).

View larger version (27K): [in this window] [in a new window]   Figure 7. A, Average force–[Ca2+] relations for control (CON) myocytes (n=6) before (CON-PP2a) and after (CON+PP2a) incubation with the catalytic subunit of PP2a (0.15 U/mL). B and C, Bar graphs of averaged curve fit parameters (Fmax and EC50) from CON cells before and after treatment with PP2a. D, Average force–[Ca2+] relations for CHF myocytes (n=7) before (CHF-PP2a) and after (CHF+PP2a) incubation with the catalytic subunit of PP2a (0.15 U/mL). E and F, Bar graphs of averaged curve fit parameters (Fmax and EC50) from CHF cells before and after treatment with PP2a. *Significant vs CHF-PP2a (P<0.05).

View larger version (15K): [in this window] [in a new window]   Figure 8. Average force–[Ca2+] relations for control (CON) myocytes (n=6) before treatment (CON-Baseline), after treatment with PKC- (CON+PKC-), and after treatment with PP1 (CON+PP1). *Significant vs CON-Baseline (P<0.05); #significant vs CON-Baseline and CON+PP1 (P<0.05).

	Discussion

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These results are the first to show that PKC- expression and activity is upregulated to a similar degree in two divergent rat models of end-stage CHF. Our observation that in nonfailing cells, PKC- induces significant depression of myofilament function, whereas in failing cells it is without a functional effect, suggests a role for PKC- in the modulation of myofilament function in both health and disease. Our finding that PP1 treatment can recover myofilament force development and Ca²⁺ sensitivity in failing cells and nonfailing cells treated with PKC- confirms that augmented phosphorylation of myofilament targets, attributable, in part, to increased PKC- signaling, plays a key role in the contractile dysfunction observed in failing ventricular myocytes. Therefore, phosphorylation of myofilament proteins mediated by PKC- may be a worthy signaling mechanism to target to restore contractile function of the ventricular myocyte in end-stage CHF.

PKC- Expression and Activation in Experimental CHF
Several PKC isozymes have been linked to ventricular remodeling secondary to myocardial infarction and pressure overload.² However, previous studies have found increased or unaltered activation and expression of PKC- in experimental models of LVH and CHF.^18,27,28 These divergent findings may relate to the duration (acute/compensated versus chronic/end-stage) and/or etiology (myocardial infarction, pressure overload, volume overload, genetic hypertension) of heart disease. For these reasons, we examined PKC- expression and activation in left ventricles obtained from rats in early HF and end-stage CHF. At the early stage, PKC- concentrations and activity were unchanged. In contrast, at end-stage CHF, we found comparable increases in PKC- content and activity in both experimental models, which is in agreement with previous work.^18,20,23 Indeed, our data suggest that PKC- plays a key role in mediating the transition to end-stage CHF and is involved in myofilament dysfunction in the rat ventricle. The role of PKC- in impaired ventricular myocyte contractility has thus far been primarily attributed to alterations in Ca²⁺ homeostasis.²³ Our results suggest that PKC-–dependent blunting of ventricular myocyte contractility is more complex and also involves direct hyperphosphorylation of myofilament proteins.

PKC-–Dependent Myofilament Protein Phosphorylation in End-Stage CHF
Myofilament dysfunction as a basis for reduced ventricular myocyte contractility has emerged as an important molecular mechanism in the failing heart.^3,29 That is, we and others have shown both in experimental and human CHF a significant blunting of myofilament force development, Ca²⁺ sensitivity, and cross-bridge cycling kinetics.^{3,11–14,16,17,25,26,29,30} In transgenic mouse models of HF, reports indicate that increased myocardial concentrations of PKC-ß and PKC- elicit augmented phosphorylation of cTnI and cTnT coincident with myofilament and ventricular dysfunction.^7,21,22 Moreover, several studies clearly indicate that phosphorylation of cTnI and cTnT by numerous PKC isozymes are central in the control of ventricular myofilament force development, Ca²⁺ sensitivity, and contractility.^4–6,9,24 For example, using reconstituted myofilament preparations, Jideama et al have shown that PKC-, PKC-ß, PKC-, and PKC- phosphorylate cTnI and cTnT at multiple sites.²⁴ Moreover, because of their propensity for cross phosphorylation of cTnI and cTnT sites, the number of phosphorylation sites, and the myriad PKC isozymes, clearly defining the relative contribution of each specific PKC isozyme in control of myofilament force development, and Ca²⁺ sensitivity is rather difficult to discern.^4,24 In addition to PKC-, we also examined protein expression of other PKC isozymes known to (1) phosphorylate cTnI and cTnT; (2) be altered in ventricular hypertrophy and failure; and (3) be predominately expressed in the nonfailing rat ventricle, namely PKC-ß, PKC-, and PKC-.³¹ We found no changes in expression of these enzymes in end-stage CHF (unpublished observations, 2005). Thus, we chose to focus on PKC- for several reasons. First, as mentioned, it was the only PKC isozyme that, in our hands, appears involved in both reduced myofilament contractility and in mediating the transition to end-stage CHF. Secondly, PKC- expression and activity are increased in end-stage human CHF of diverse etiology, thus making it a clinically relevant molecular target.^19,30 Lastly, PKC- translocates to the cardiac sarcomere following heightened neurohormonal stimulation³² and blunts myofilament function.^4,5,24

Until now, data were scant regarding PKC-–dependent signaling to myofilaments in chronically failing myocardium. Importantly, we have recently demonstrated that alterations in cardiac troponin, most likely phosphorylation, cause myofilament dysfunction in our CHF models. In skinned ventricular myocytes, we found that PKC-–induced phosphorylation had no impact on myofilament mechanics in failing cells but resulted in a significant depression of Ca²⁺-saturated force and myofilament Ca²⁺ sensitivity in control cells. Furthermore, we also found that PP1-dependent dephosphorylation could rescue the depressed myofilament phenotype in control myofilaments hyperphosphorylated with PKC-. Taken together, these findings confirm that heightened PKC- signaling contributes to myofilament dysfunction in end-stage CHF. In agreement with our findings, Noguchi et al also found increased activation of PKC- in parallel with reduced thin-filament function in reconstituted myofilaments prepared from failing human hearts.³⁰ Moreover, in that study, normalization of PKC- activity by mechanical unloading prompted a restoration of maximal force development in failing myofilaments. Hence, our findings in animal CHF may also be applicable to human end-stage CHF. In contrast, recent reports show a decline in myofilament Ca²⁺ sensitivity after treatment with the catalytic subunit of PKC in diseased or nondiseased human and porcine skinned myocytes but to a greater extent in failing myocardium.^17,33 Those results suggest a reduction in PKC-mediated phosphorylation of failing myofilaments. These investigators also found no effect of PKC treatment on maximal Ca²⁺-saturated force development. The inconsistencies may relate, in part, to the use of human tissue where significant catecholamine signaling in donor myocardium may have occurred.^34,35 Furthermore, because those studies relied on the use of the catalytic subfragment of PKC, it is plausible that reagent induced promiscuous phosphorylation of other myofilament proteins (eg, cTnI, cTnT, myosin light chain-2, and myosin-binding protein C) occurred, making these data difficult to interpret. Here, we avoided this potentially confounding factor by examining the direct functional impact of PKC-; the specific isoform that we found upregulated in CHF. In reconstituted systems and multicellular preparations, PKC- has been shown to phosphorylate serines 43 and 45 on cTnI and cTnT, which diminishes myofilament contractility.²⁴ Moreover, Sumandea et al showed that PKC-–dependent phosphorylation of cTnT at the threonine 206 residue was both necessary and sufficient to induce depressed myofilament function.⁵ Collectively, these data suggest a specific role for PKC- in both failing and nonfailing myofilament function. However, further studies are required to identify which sites on cTnI and cTnT undergo PKC-–dependent phosphorylation in failing myofilaments and are the specific sites that contribute to ventricular myocyte contractile dysfunction. Overall, though, there are considerable data that PKC induces ventricular myocyte dysfunction by hyperphosphorylation of the myofilaments; studies also suggest that PKC isozymes may elicit contractile dysfunction by altering (1) calcium handling/L-type Ca²⁺ channel function, (2) cytoskeletal function, and (3) coupling through the ß-adrenergic receptor.^2,23,36

Protein Phosphatase Expression and Regulation of Myofilament Function
In the mammalian ventricular myocyte, PP1 and PP2a account for the majority (>90%) of the phosphatase activity.³⁷ Some studies suggest that PP1 activity and expression is increased in end-stage CHF.³⁸ Additionally, transgenic overexpression of PP1 and PP2a results in cardiac hypertrophy, contractile dysfunction, altered phosphorylation of phospholamban, and cTnI, along with progression to end-stage CHF in murine models.^39,40 Thus, it seems plausible that altered expression and activity of either PP1 or PP2a may explain the ventricular dysfunction observed in end-stage CHF. However, we found no change in expression of either protein phosphatase, suggesting that the reduced myofilament phenotype is not attributable to altered expression of PP1 or PP2a in the failing rat ventricle.

If augmented myofilament protein phosphorylation underlies depressed myofilament function in the failing myocyte, then dephosphorylation by protein phosphatases would be expected to improve myofilament function in CHF. Indeed, we observed that PP1-dependent dephosphorylation of failing myofilament proteins improved myofilament function in failing cells but not in control cells. A recent study using human skinned myocytes also found improved function following PP1 treatment, although these investigators attributed this to dephosphorylation of myosin light chain-2. It has been suggested that PP1 predominantly modulates Ca²⁺ homeostasis by dephosphorylating phospholamban. However, PP1 is also docked at the myofilament and thus may modulate myofilament protein phosphorylation.⁴¹ PP2a has been suggested to be specific for dephosphorylation of cTnI at Ser23 and -24 sites. However, we found that PP2a treatment in either skinned failing or control myocytes was without a significant effect on myofilament function. Recent work by Chen et al shows that PP2a dephosphorylates MLC-2 in skinned ventricular myocytes. Thus, it is possible that in addition to dephosphorylating cTnI, PP2a also dephosphorylates MLC-2. It may be that the cumulative impact of these functionally opposing dephosphorylation events leaves myofilament function relatively unaltered. On the other hand, in reconstituted failing myofilaments from human ventricles, Noguchi et al, found that PP2a treatment caused an increase in maximal force development and a decrease in maximal sliding velocity.³⁰ It is conceivable, therefore, that PP2a is more effective in human cardiac muscle; such a finding would imply important species differences; further investigation is required to resolve this issue. Our observation that PP1-induced dephosphorylation restores myofilament function in failing myocytes and control myocytes treated with PKC- clearly suggests that augmented phosphorylation of myofilament constituents plays an important role in depressed myofilament function in end-stage CHF.

In summary, we have identified PKC- as an important signaling molecule that converges onto the myofilament lattice to induce hyperphosphorylation and consequent depression of ventricular myofilament force development, Ca²⁺ sensitivity, and contractility in different models of end-stage CHF. Inhibition of PKC- activity may be a novel therapeutic route through which ventricular myofilament contractility could be restored in the setting of CHF.

	Acknowledgments

 
We thank David L. Geenen, Dalia Urbonini, and Milana Yuzhakova for assistance in generating the experimental models.

Sources of Funding

This work was supported by NIH grants HL64035, HL77195, HL62426, and T32-007692 and American Heart Association Grants 0335199N and 0230038N. R.J.B. was supported by a United Negro College Fund–MERCK Predoctoral Fellowship and an American Physiological Society Porter Physiology Fellowship.

Disclosures

None.

	Footnotes

 
Original received January 15, 2007; revision received May 3, 2007; accepted May 24, 2007.

	References

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