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Am J Physiol Heart Circ Physiol 291: H2344-H2353, 2006. First published June 30, 2006; doi:10.1152/ajpheart.00541.2006
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Left ventricular myofilament dysfunction in rat experimental hypertrophy and congestive heart failure

Rashad J. Belin,1 Marius P. Sumandea,1 Tomoyoshi Kobayashi,1 Lori A. Walker,1,2 Veronica L. Rundell,1,2 Dalia Urboniene,2 Milana Yuzhakova,2 Stuart H. Ruch,1,2 David L. Geenen,2 R. John Solaro,1 and Pieter P. de Tombe1,2

Center for Cardiovascular Research, 1Department of Physiology and Biophysics, Department of Medicine; and 2Section of Cardiology, University of Illinois, Chicago, Illinois

Submitted 26 May 2006 ; accepted in final form 28 June 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
It is currently unclear whether left ventricular (LV) myofilament function is depressed in experimental LV hypertrophy (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. A subset of cells was treated with either unphosphorylated, recombinant cardiac troponin (cTn) or cTn purified from either control or failing ventricles. LV myofilament function was characterized by the force-[Ca2+] relation yielding Ca2+-saturated maximal force (Fmax), myofilament Ca2+ sensitivity (EC50), and cooperativity (Hill coefficient, nH) parameters. POLVH was associated with a 35% reduction in Fmax and 36% increase in EC50. Similarly, MICHF resulted in a 42% reduction in Fmax and a 30% increase in EC50. Incorporation of recombinant cTn or purified control cTn into failing cells restored myofilament Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ (increased Ca2+ 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 phosphorylation of cardiac troponin (cTn) I (cTnI), and cTnT induces depressed myofilament function in reconstituted myofilaments and multicellular preparations (2, 37). Moreover, recent in vitro motility studies revealed thin filament dysfunction in failing human hearts secondary to altered Tn phosphorylation (17). Therefore, we postulated that functionally important alterations in cTn contribute to depressed contractile function in experimental LVH and CHF. Accordingly, our aims here were the following: 1) determine whether myofilament function in LV myocytes is depressed in experimental LVH and CHF, 2) determine whether alterations in cTn contribute to myofilament dysfunction, and 3) examine the phosphorylation profile of cTnI in failing ventricles. To address these aims, two rat models were used that undergo different modes of ventricular remodeling: pressure overload-induced LV hypertrophy (POLVH) and myocardial infarction (MI)-elicited congestive heart failure (MICHF) (3, 4, 16, 25, 29, 43). Myofilament function was determined in skinned LV myocytes, and the role of cTnI was assessed by in vitro exchange of endogenous Tn with exogenous (recombinant or tissue purified) cTn and analysis of cTnI phosphorylation via one-dimensional isoelectric focusing analysis.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal models of experimental LVH and CHF. Ascending aortic banding was performed on 4-wk-old female Sprague-Dawley rats as described previously with slight modifications (16). MI was induced in 4-wk-old female Sprague-Dawley rats as described earlier (10). Animals were followed for a period of 32–36 wk until they transitioned to a stage of POLVH and MICHF. Unoperated age-matched animals served as controls. Previously, we found no difference between sham-operated and age-matched control animals (9, 10). LV hemodynamic function was determined as described in detail earlier (10). After the animal was euthanized, micrometry was utilized to determine ventricular dimensions.

Force-[Ca2+] measurements in skinned ventricular myocytes. Myocytes were isolated from the interventricular septum and LV free wall of MICHF, POLVH, and age-matched control hearts by mechanical homogenization and chemically permeabilized (skinned) with 0.3% Triton X-100 (15, 20). We observed no difference in myofilament function between septal and LV myocytes (data not shown). Details of the solutions for cell isolation and experimentation have been previously described in detail (9, 15, 20). Cells were stored on ice and used within 20 h of isolation. All single cell experiments were performed on the stage of an inverted microscope as previously described (9, 15, 20). Sarcomere length was set to 2.10 µm by video micrometry (Fig. 1) (9, 15).


Figure 1
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  		Fig. 1. A: photomicrograph of an attached cardiac myocyte in relaxing solution at sarcomere length = 2.10 µm. B: side view illustrating direct measurement of myocyte height in relaxing solution at sarcomere length = 2.10 µm.

 
Isolation of cTn. For preparation of bacterially expressed cTn, we used a similar protocol as described previously (37). Whole cTn was extracted and purified from MICHF and control ventricles as described previously with minor modifications (19, 34). To preserve endogenous myofilament protein phosphorylation status, all solutions contained okadaic acid, calyculin A, and protein kinase inhibitor cocktail (Sigma Aldrich).

Troponin exchange into skinned myocytes. Skinned cardiac myocytes from POLVH, MICHF, and age-matched control ventricles were washed with Tn exchange buffer and then resuspended in either relaxing solution or Tn exchange buffer containing either recombinant (rTnEx) or purified (pTnEx) cTn. Skinned cells were incubated (exchanged) overnight at 4°C. To accurately quantify the extent of in vitro replacement of endogenous cTn with unphosphorylated, recombinant cTn in cardiac myocytes, we performed a serial dilution immunoassay (42). The recombinant cTnT contained a nine amino acid Myc tag at the NH2-terminus that slows its migration through the gel and can thus be used to visualize replacement of endogenous TnT following the rTnEx protocol (37). In seven independent experiments, we found that the degree of rTnEx was 70 ± 2.8.

Determination of cTnI phosphorylation profile. One-dimensional, nonequilibrium, isoelectric focusing (1D-IEF) analysis of cTnI-phosphorylated species was executed as described earlier (18, 35). Briefly, ventricular myocardium frozen at –80°C was homogenized in buffer containing 8 M urea, 2.5 M thiourea, 4% CHAPS, 0.5% ampholytes (3–10), 2 mM EDTA, and 2 mM tributylphosphine and protease inhibitor cocktail (Sigma Aldrich). The homogenized samples were then separated on IEF gels, which contained 8 M urea, 5% acrylamide (acrylamide/bis-acrylamide 29:1) 2% Triton X-100, 0.8% ampholyte (pH 3.5–10.0) and 1.2% ampholyte (pH 7.0–9.0) (Amersham, NJ). The sample loading buffer contained 8 M urea, 10 mM EDTA, and 1.0% ampholyte (pH 3.5–10.0). Both urea and EDTA assisted with the dissociation of cTnI from other subunits within the cTn complex. The upper and lower reservoirs were filled with 0.01 H3PO4 (cathode buffer) and 0.02 M NaOH (anode buffer), respectively. The positive and negative ports were placed into the negative and positive ports, respectively. Gel electrophoresis was executed at 100 V for 30 min, 200 V for 30 min, and 500 V for 10 min without prerun. For the immunoblot, after separation, proteins were transferred to nitrocellulose membranes overnight at 30 V at 4°C. Membranes were blocked for 1 h in 2% nonfat dry milk in Tris-buffered saline with Tween 20 (TBST), washed and incubated with monoclonal anti-cTnI antibody (1:2,500 in TBST; clone C5 from Research Diagnostics) for 3 h at room temperature. After being washed with TBST, the blots were incubated with anti-mouse secondary antibody linked to horseradish peroxidase. Proteins were visualized by using enhanced chemiluminescence (Amersham). Films were scanned and cTnI-phosphorylated species were quantified by densitometric analysis using Image J. TnI phosphorylation is expressed as %phosphorylated = TnI (phosphorylated)/TnI (phosphorylated + unphosphorylated) x 100%. All samples were run in triplicate.

Data and statistical analysis. The force-[Ca2+] relation was fit to a modified Hill equation: F = Fmax x [Ca2+]nH/([Ca2+]nH+ EC50nH), where F is developed force (mN/mm2), Fmax is the force at maximal calcium activation, EC50 is the [Ca2+] at 50% maximal activation and represents the myofilament Ca2+-sensitivity index, and nH represents the slope of the force-[Ca2+] relation (Hill coefficient). In some cases, data obtained from two or more myocytes from each heart were averaged and represented a single measurement value. Differences between myocyte data from POLVH, MICHF, and age-matched controls were determined by general linear model analysis of variance (SPSS; version 11). Data are expressed as means ± SE. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Impact of POLVH and MICHF on cardiac morphology. POLVH and MICHF resulted in different modes of morphological remodeling (Tables 1 and 2). The cardiac myocyte isolation technique precluded direct assessment of LVH; however, indexes of whole heart (heart weight-to-body weight ratio, HW:BW) and RV (RV weight-body weight ratio, RVW:BW) hypertrophy were measured. When compared with age-matched controls, POLVH resulted in a 130% increase in HW:BW and a 118% increase in RVW:BW, whereas MICHF resulted in a 34% increase in HW:BW and 91% increase in RVW:BW. Additionally, LV wall thickness was increased by 45% in POLVH hearts, and IVS wall thickness was increased by 31% in MICHF hearts. MICHF was associated with a 100% increase in transverse LV luminal diameter, whereas POLVH resulted in a less severe dilatation (31% increase). Also, both POLVH and MICHF resulted in pulmonary congestion, pleural effusion, and, several weeks before animal euthanasia, increased mortality. Thus both models progressed to end-stage CHF. Our results parallel those described recently by others who also employed a chronic infarction rat model of CHF (36). Myocyte morphology was assessed at a sarcomere length of 2.10 µm (Fig. 1). Two-way analysis of variance revealed that myocytes from both models exhibited comparable increases in length, width, height, and cross-sectional area relative to control cells.


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  		Table 1. Morphological and hemodynamic characteristics: pressure overload-elicited left ventricular hypertrophy

 

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  		Table 2. Morphological and hemodynamic characteristics: myocardial infarction-induced congestive heart failure

 
Effect of POLVH and MICHF on LV pump function. Baseline LV hemodynamics are summarized for POLVH in Table 1 and MICHF in Table 2. LV systolic pressure development was preserved in POLVH as shown by a 25% increase in peak LVSP and no change in LV developed pressure. In contrast, LV systolic function was impaired in MICHF as indexed by a 22% reduction in LV developed pressure and no change in LV systolic pressure. Both models exhibited marked elevation in LV end-diastolic pressure. However, the degree of diastolic dysfunction was greater in POLVH where LV end-diastolic pressure increased by 600% in contrast to the 300% increase in MICHF hearts. Significant reductions in derived indexes of the rate of pressure rise and decline were observed in both animal models.

Impact of POLVH and MICHF on LV myofilament function. A total of 23 myocytes were studied from 10 POLVH hearts and 19 myocytes from 10 age-matched control hearts. Figure 2 illustrates the impact of POLVH on LV myofilament function. When compared with age-matched controls, POLVH myocytes displayed a 35% reduction in Ca2+-saturated Fmax and a 36% increase in the EC50 parameter (myofilament Ca2+-sensitivity index). The slope of the force-[Ca2+] relation, the Hill coefficient (myofilament cooperativity index), was unaltered by POLVH. Figure 2 also illustrates the impact of MICHF on LV myofilament function. Twenty-three myocytes were studied from 15 MICHF hearts and 20 myocytes from 15 age-matched control hearts. MICHF resulted in a 42% decrease in the Fmax, a 30% increase in the EC50, and no change in the Hill coefficient. Finally, passive force in relaxed skinned myocytes was reduced by 35% in POLVH and 46% in MICHF relative to their respective controls at a sarcomere length of 2.10 µm. Two-way analysis of variance revealed that both POLVH and MICHF induced an identical and severe blunting of LV myofilament function (Table 3). Therefore, despite the diverse etiology of cardiac disease in these two rodent models, the end impact on LV myofilament mechanics was virtually identical.


Figure 2
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  		Fig. 2. A: average force-[Ca2+] relations for pressure overload-induced left ventricular hypertrophy (POLVH), myocardial infarction-elicited congestive heart failure (MICHF), and age-matched control myocytes. Data were obtained from 19 myocytes from 10 control (CON)-POLVH animals, 20 myocytes from 15 CON-MICHF animals, 23 myocytes from 10 POLVH animals, and 23 myocytes from 15 MICHF animals. B and C: averaged curve-fit parameters {maximal Ca2+-saturated force development (Fmax), and [Ca2+] at 50% of Fmax (EC50)}. *P < 0.05 vs. CON-POLVH, CON-MICHF.

 

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  		Table 3. Impact of POLVH and MICHF on LV myofilament function in skinned cardiac myocytes

 
Recombinant TnEx restores LV myofilament function in POLVH and MICHF myocytes. To test the hypothesis that functionally important alterations in cTn underlie depressed LV myofilament function in experimental LVH and CHF, we employed in vitro rTnEx in POLVH, MICHF, and age-matched control myocytes. Figure 3 illustrates the effect of rTnEx on myofilament function in POLVH and age-matched control cells. In control cells, rTnEx was without a significant effect on myofilament Ca2+ sensitivity (indexed by the EC50 parameter). In contrast, rTnEx in POLVH cells induced a 29% decrease in the EC50 (P < 0.05) relative to untreated POLVH cells. Hence, rTnEx restored myofilament Ca2+ sensitivity to levels observed in nonfailing control cells. The impact of rTnEx in MICHF and age-matched control cells is shown in Figure 4. Again, rTnEx in control cells resulted in little, if any, change in the EC50 parameter, whereas incubation of MICHF myocytes with recombinant cTn resulted in a 33% decline in the EC50 (P < 0.05). Maximum force development, on the other hand, was not markedly affected by Tn exchange. To our knowledge, this is the first report to examine the structure-function relation of cTn in mechanically isolated, skinned cardiac myocytes using in vitro Tn exchange. Previous studies from our group and others have used skinned multicellular fibers. Generally, investigators reported a near-complete replacement of endogenous cTn in such preparations (2, 37). In batches of mechanically isolated, skinned myocytes and myofragments, we found that 70% of the endogenous cTn was displaced. It may be that differences in myocardial preparation account for the divergence in Tn exchange between previous work and studies reported here. In our study, mechanical homogenization was used to yield cells from ventricular biopsies. As a result, in addition to cardiac myocytes, we frequently obtained larger myofragments that consist of many undissociated cardiac myocytes that, when immersed in Tn exchange buffer, are not completely exchanged. Subsequently, these unexchanged myofragments may have been solubilized in sample buffer and run on gels without much of the recombinant cTn being integrated into the myofilament lattice. The net effect would be a "dilution" of the proportion of cTn exchange, which occurred in these cell batches. Consequently, in the single skinned cells, used for mechanical analyses, we expect that Tn exchange percentage was greater. Nevertheless, our data clearly demonstrate that depressed LV myofilament Ca2+ sensitivity in ventricular cells from two different rodent models of end-stage cardiac disease is due to functionally important alterations in the cTn complex.


Figure 3
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  		Fig. 3. A: average force-[Ca2+] relations for CON myocytes incubated with recombinant cardiac troponin (cTn) (CON-rTnEx) or relaxing solution (CON). Data were obtained from 5 treated ({triangleup}) and 5 untreated ({square}) myocytes gathered from 5 control hearts. Fmax was 16.9 ± 1.2 and 20.4 ± 1.7 mN/mm2 for treated and untreated cells, respectively (P > 0.05). B: average force-[Ca2+] relations for POLVH myocytes exposed to recombinant cTn (POLVH-rTnEx) or relaxing solution (POLVH). Data were obtained from 7 treated ({triangleup}) and 7 untreated ({square}) myocytes gathered from 7 POLVH hearts. Fmax was 13.0 ± 1.6 and 13.0 ± 1.0 mN/mm2 for treated and untreated cells, respectively (P > 0.05). C: EC50 values from CON and POLVH myocytes with and without recombinant cTn exposure. *P < 0.05 vs. CON, CON-rTnEx. #P < 0.05 vs. POLVH.

 

Figure 4
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  		Fig. 4. A: average force-[Ca2+] relations for CON cells incubated with recombinant cTn (CON-rTnEx) or relaxing solution (CON). Data were obtained from 15 treated control cells from 12 hearts ({circ}) and 8 untreated control myocytes from 8 hearts ({square}). Fmax was 16.7 ± 1.4 and 21.3 ± 1.6 mN/mm2 for treated and untreated cells, respectively (P > 0.05). B: average force-[Ca2+] relations for MICHF myocytes treated with recombinant cTn (MICHF-rTnEx) or relaxing solution (MICHF). Data were obtained from 16 treated cells from 12 MICHF hearts ({circ}) and 7 untreated cells from 7 MICHF hearts ({square}). Fmax was 12.3 ± 1.3 and 12.1 ± 1.5 mN/mm2 for treated and untreated cells, respectively (P > 0.05). C: EC50 values from CON and MICHF myocytes with and without recombinant cTn exposure. *P < 0.05 vs. CON, CON-rTnEx. #P < 0.05 vs. MICHF.

 
Impact of purified TnEx on LV myofilament function in control and failing cardiac myocytes. To further test the hypothesis that Tn contributes to myofilament dysfunction in experimental CHF, we purified cTn from nondiseased and MICHF left ventricles. Figure 5, A and C, illustrates the effect of cTn purified from control or MICHF hearts, respectively, on LV myofilament function in MICHF cells. As was the case with rTnEx, treatment of MICHF cells with purified control cTn induced a 22% decline in EC50 (P < 0.05) relative to untreated failing cells. In contrast, failing cTn integrated into MICHF cells elicited no significant effect. These findings indicate that reduced myofilament Ca2+ sensitivity in failing myocytes can be restored by purified, nondiseased cTn. Figure 5, B and D, also shows the reverse experiment, that is, the effect of exchange of purified control or failing cTn, respectively, on LV myofilament function in control cells. Incorporation of control cTn into control cells had no effect on Fmax development and the EC50, whereas failing cTn exchanged into control cardiac myofilaments resulted in a 27% increase in EC50 (decreased Ca2+ sensitivity) (P < 0.05) and a nonsignificant (P = 0.10) reduction in Fmax development. Thus integration of failing cTn into control cells induced a severe depression of LV myofilament Ca2+ sensitivity. These results lend further support to the notion that functionally important alterations in cTn contribute to reduce LV myofilament Ca2+ sensitivity in experimental CHF.


Figure 5
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  		Fig. 5. A: average force-[Ca2+] relations for MICHF myocytes (n = 12), MICHF myocytes treated with purified, control cTn [MICHF-pTnEx (CON)] (n = 8), and purified, failing cTn [MICHF-pTnEx (CHF)] (n = 4). A total of 8 hearts were used to obtain the cell data. Fmax was 12.6 ± 1.1, 13.2 ± 1.5, and 12.4 ± 1.7 mN/mm2 for MICHF, MICHF-pTnEx (CON), and MICHF-pTnEx (CHF) cells, respectively, (P > 0.05). B: average force-[Ca2+] relations for CON (n = 6), CON cells treated with purified, control cTn [CON-pTnEx (CON)] (n = 11), and purified, failing cTn [CON-pTnEx (CHF)] (n = 11). A total of 9 hearts were used to obtain the cell data. Fmax was 19.9 ± .94, 19.5 ± 1.6, and 14.8 ± 1.4 mN/mm2 for CON, CON-pTnEx (CON), and CON-pTnEx (CHF) cells, respectively, (P > 0.05). C: EC50 values for MICHF, MICHF-pTnEx (CON), and MICHF-pTnEx (CHF) myocytes. *P < 0.05 vs. MICHF. D: EC50 values for CON, CON-pTnEx (CON), and CON-pTnEx (CHF) myocytes. *P < 0.05 vs. CON and CON-pTnEx (CON).

 
cTnI phosphorylation in experimental LVH and CHF. Figure 6A illustrates representative one-dimensional isoelectric focusing gels revealing the phosphorylation profile of cTnI in control, LVH, and CHF left ventricles. The total proportion of phosphorylated cTnI in control samples was 75% and increased to 86% in POLVH and MICHF left ventricles (Fig. 6B). We also observed four phospho-states of cTnI in control ventricular homogenates where the proportion of cTnI in the highest phosphorylation state (P') was ~3%, whereas 7 cTnI phospho-states were observed in POLVH and MICHF left ventricles and the proportion of cTnI in the P' state increased substantially to ~20% (Fig. 6C). Overall, these results suggest that in addition to an increase in total cTnI phosphorylation, there is also a redistribution of phosphoryl groups along cTnI such that additional sites become phosphorylated in end-stage experimental CHF.


Figure 6
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  		Fig. 6. A: representative immunoblots of cTnI phosphospecies in CON (n = 8), POLVH (n = 8), and MICHF (n = 8) ventricular samples separated by nonequilibrium isoelectric focusing as described previously (1, 35). A detailed description is also provided in METHODS. Each lane was evenly loaded with 25 µg of total protein. cTnI phosphospecies were detected with a monoclonal anti-cTnI antibody (1:2,500 in TBST; clone C5 from Research Diagnostics). All samples were run in triplicate, and cTnI phosphorylation was determined by an average of the three gels. Shown are the unphosphorylated (U) and highest phosphorylated (P') states of cTnI. cTnI indicates recombinant, unphosphorylated cTnI. B: histograms summarizing the percentage of cTnI phosphorylation determined ratiometrically as follows: %phosphorylated cTnI = TnI (phosphorylated)/TnI (phosphorylated + unphosphorylated) x 100%. Phosphorylated cTnI includes all the bands above the lowest (unphosphorylated) band. C: proportion of cTnI that exists in the highest (P') phosphorylated state. *Considered significant relative to CON (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In work delineated here, we are the first to demonstrate that, in the rat, LV myofilament function is depressed in both POLVH and MICHF to a strikingly similar degree. Furthermore, exchange with either recombinant, unphosphorylated cTn or cTn purified from control hearts restored LV myofilament Ca2+ sensitivity in failing cells to control levels. In contrast, exchange with purified failing cTn into control cells induced a marked reduction in LV myofilament Ca2+ sensitivity. One-dimensional nonequilibrium isoelectric focusing analysis revealed a slight, but significant, increase in total cTnI phosphorylation in both LVH and CHF ventricles. Collectively, these findings indicate that depressed LV myofilament Ca2+ sensitivity in experimental LVH and CHF is due, in part, to functionally important alterations in the cTn complex, which involve augmented phosphorylation of cTnI.

Animal models of experimental LVH and CHF. Consistent with previous results, pressure overload resulted in marked cardiac and RV hypertrophy, increased LV wall thickness, increased transverse LV diameter, preserved systolic pressure development, and marked diastolic dysfunction (Table 1) (9, 16, 24). In contrast, a large LV myocardial infarction resulted in moderate cardiac and RV hypertrophy, increased septal wall thickness, increased transverse LV diameter, eccentric LVH, blunted systolic function, and diastolic dysfunction, as previously reported (Table 2) (4, 5, 7, 11, 22, 29, 43, 46). Whereas the specific and selective molecular mediators of the divergent hypertrophic responses have not been clearly defined, there is evidence to suggest that different biochemical and mechanical signals invoked by pressure overload and myocardial infarction underlie the distinct morphological and functional remodeling that characterize these etiologies of heart failure (3, 46). Indeed, in our rodent models, these signals led to two divergent phenotypes of cardiac dysfunction: predominantly diastolic heart failure with preserved systolic pressure development (POLVH) and systolic heart failure with mild diastolic dysfunction (MICHF).

LV myofilament function in experimental LVH and CHF. Myofilament function in animal models of LVH and CHF has thus far been reported to be either unchanged, increased, or decreased (5, 6, 14). This lack of consensus may be due to: 1) the duration of cardiac disease (compensated LVH vs. end-stage CHF), 2) the etiology of cardiac disease, 3) the myocardial preparation utilized (e.g., cells vs. fiber bundles), and 4) chamber-specific variation in myofilament function (RV vs. LV). We have previously shown that 12 wk after MI in the rat, a time point at which no observable clinical features of CHF are manifest, myofilament function is unchanged (4). However, 24 wk after MI, with the transition to overt CHF in this model, myofilament function was severely blunted (7). Similar observations have also been made in the aortic-banded rat model of LVH (16, 24). Studies using multicellular isolated myocardium are somewhat limited; however, though such preparations are metabolically stable and structurally ideal for measurement and control of sarcomere length (4, 7), they consist of many cells embedded in an intact and possibly stiffer collagen matrix. It is well documented that the extracellular matrix changes markedly in LVH and CHF (5, 6, 14). Moreover, it is expected that collagen matrix expansion would decrease the number of functional myofilaments per cross-sectional area, which would manifest as depressed maximum force-generating capacity. Hence, studies in single cells are preferable because they allow for a more direct and accurate assessment of alterations in myofilament function. Several investigators (11, 22, 25, 32) have shown that both pressure overload and myocardial infarction of the LV induce changes in myofilament architecture that are not uniformly distributed to the neighboring right ventricle. To overcome these limitations, we chose here to study single skinned myocytes mechanically isolated from the left ventricle. By employing two chronic rat models of end-stage CHF, as confirmed by invasive hemodynamic and post mortem morphological analyses, we observed a marked and strikingly similar reduction in LV myofilament function both in terms of Fmax development and myofilament Ca2+ sensitivity. Hence, we conclude that ventricular dysfunction in these models of end-stage cardiac disease has, at its basis, a reduction in myofilament function.

The current myofilament mechanical findings are consistent with our previous findings in the right ventricle (7, 9) and, more recently, in the human diabetic left ventricle (15). A decrease in Fmax development has also recently been found in a pig myocardial infarction model (41). Specifically, we observed a 38% reduction in Fmax and 32% increase in the EC50 in diseased LV cells, which parallels the 35% reduction in Fmax and 31% increase in the EC50 that we reported in hypertrophied RV myocytes (9). Though myocytes in our study generated less force than previous work, comparison of our study in single cells with earlier work using multicellular preparations should be made with caution because several reports indicate that skinned trabeculae consistently generate roughly twice the Fmax as cardiac myocytes (7, 9, 15, 20, 31, 37, 40, 41, 44, 45). The differences in Fmax development between single and multicellular preparations may relate to the accuracy with which the cross-sectional area of the muscle preparation was determined. Despite this, Fmax in control cells in this and previous work was ~20 mN/mm2 (15, 41). Furthermore, diseased cells from all studies manifested a marked blunting of the Fmax. In contrast, Wolff et al. (45) found that in canines, CHF myofilament Ca2+ sensitivity is increased, and similar observations have also been made in human CHF (40, 44). Importantly, cells obtained from the canine pacing model did not manifest the changes in cellular morphology expected from remodeling processes invoked by chronic hemodynamic stress. Moreover, treatment with protein kinase A in those studies induced a marked decrease in myofilament Ca2+ sensitivity in the cardiac disease group, but not the control group, suggesting that protein kinase A-mediated phosphorylation of contractile proteins was already maximal in that group of cells (40, 44, 45). In our study, employing two well-defined models of end-stage cardiomyopathy in the rat, on the other hand, cardiac disease was associated with an increase as opposed to decreased level of contractile protein phosphorylation (Fig. 6 and see Molecular mechanisms of myofilament dysfunction). Thus differences in the etiology and duration of cardiac disease, species, or tissue handling procedures may account for the disparate findings regarding myofilament Ca2+ sensitivity in the various studies.

Molecular mechanisms of myofilament dysfunction. In the thin filament, the cTn operates as the molecular switch for muscle contraction and is composed of three distinct gene products: cTnC, which is the Ca2+ receptor; cTnI, which binds actin and structurally inhibits the actin-myosin interaction; and cTnT, which tethers cTn within the thin filament via its binding to tropomyosin (6, 37). Calcium binding to Tn leads to activation of the thin filament, binding of myosin, the formation of active cycling cross-bridges, and ultimately myocyte force development and sarcomere shortening (6, 37). Depressed myofilament function, as seen in the present study, may thus be caused by alterations, either pre- or posttranslational, in any of these contractile protein systems. In particular, such alterations include oxidation, proteolysis, isoform shifts, and phosphorylation. For example, recent studies (8) suggest that there is augmented oxidation of myofilament proteins in murine cardiomyopathy. Furthermore, it is possible that cTn becomes considerably more susceptible to proteolysis in end-stage cardiac disease (23). However, such analyses are beyond the scope of investigation reported herein and warrant future study. It has been reported that reexpression of the fetal isoform of cTnT is associated with heart failure (1). However, SDS-PAGE analysis revealed no observable differential expression of cTnT in the present study (results not shown). Furthermore, recent biophysical data suggest no impact of fetal TnT on myofilament Ca2+ sensitivity (39). Likewise, although cardiac disease in small rodents is associated with increased levels of beta-myosin, this, too, does not affect the force-Ca2+ relationship in rat myofilaments (7). Additionally, we do not anticipate that the beta-myosin isoform expressed in the failing rat ventricle generates less force than the {alpha}-myosin isozyme. Indeed, by employing optical trap measurements, Sugiura et al. (36) found no difference in average force output between {alpha}- and beta-myosin isozymes. These results are also in agreement with our previous report in skinned multicellular fibers from the rat (7). Recent reports also indicate that, in heart failure, there is expression of a larger isoform of titin, which may cause a reduction in passive sarcomere stiffness (26). Our current finding of reduced passive tension in relaxed skinned myocytes in heart failure is consistent with this observation. It should be noted that in vitro Tn exchange did not affect maximum Ca2+-saturated force development. The reduced myofilament force-generating capacity seen in these small rodent models of heart failure, therefore, must have arisen from structural alterations in other contractile proteins, for example, myosin binding protein C, myosin light chain, tropomyosin, or titin. The precise molecular mechanisms underlying reduced Fmax development, however, is beyond the scope of the present study and requires further study.

The present observation that recombinant (unphosphorylated) or purified control cTn restores myofilament Ca2+ sensitivity in diseased myocytes, as well as the reverse finding that exchange of failing cTn in control cells induced a marked depression of myofilament Ca2+ sensitivity, strongly suggests a functional role for cTn in this phenomenon. Previous reports have documented that, in response to cardiac disease, there is an elaboration of mechanical and biochemical stress signals that ultimately converge to increase protein kinase C expression and activity (6, 13, 43). Furthermore, we have recently shown that protein kinase C-mediated phosphorylation of cTnI and cTnT in vitro depresses myofilament activation (2, 37). Thus it is plausible that reduced cTn function in experimental LVH and CHF arises secondary to increased phosphorylation of cTnI and/or cTnT. Several reports provide evidence to support this postulate. In the acutely infarcted rat ventricle, myofilament Ca2+ sensitivity was reduced coincident with increased cTnI and cTnT phosphorylation (21). Also, Noguchi et al. (27) showed that cTn isolated from failing rat hearts induced a depression in Ca2+ sensitivity in reconstituted myofilaments, which was normalized by endothelin receptor antagonism. In the transgenic mouse, which overexpressed PKCbeta2 in the myocardium, Takeishi and coworkers (38) illustrated that myocyte contractile function was markedly blunted secondary to depressed myofilament function and augmented phosphorylation of cTnI. Superfusion of the cells with a specific inhibitor of PKCbeta2 restored cellular contractility to normal levels. Recently, in a comprehensive and temporal analysis of the PKC{epsilon} transgenic mouse, Goldspink et al. (12) have shown that chronic PKC{epsilon} upregulation results in increased phosphorylation of cTnI and cTnT, depressed myofilament force development, and reduced ventricular pump function. Taken together, these data indicate that PKC-dependent phosphorylation of cTnI is a key modulator of myofilament function, and, consequently, ventricular pump dynamics in both health and disease.

Until now, however, it was unknown whether augmented cTnI phosphorylation contributes to myofilament dysfunction in end-stage rat CHF. Here, we found increased cTnI phosphorylation in chronically remodeled LVH and CHF left ventricles concomitant with a redistribution of phosphates on cTnI and reduced myofilament function. In particular, we report that a majority of the cTnI exists in a phosphorylated state in which multiple sites on cTnI are phosphorylated in both nonfailing and failing rodent myocardium. Interestingly, additional cTnI phospospecies became evident in CHF. These findings parallel previous work from our group in studies examining cTnI phosphorylation in the transgenic PKC{epsilon} mouse model of cardiomyopathy (35). Scruggs et al. (35) reported that a majority (nearly 75%) of the cTnI in nonfailed (nontransgenic) ventricles exists in a phosphorylated state consisting of at least two cTnI phospho-species. They suggest that the lower bands, termed P2 and P4 by the investigators, consist primarily of cTnI phosphorylated at serine-23 and/or -24. In their animal model of heart failure, they also observed a total of eight cTnI phospho-states; some of which were absent in nonfailing ventricular homogenates. Thus in two different animal models of cardiac disease, we and others (35) have found that there is incorporation of phosphate at sites on cTnI not normally phosphorylated in nondiseased myocardium. Previous work indicates that there are at least five sites within cTnI that, when phosphorylated, modulate myofilament force development, calcium sensitivity, and contractility, including Ser23, Ser24, Ser43, Ser45, and Thr144 (2, 6). It is plausible that the additional bands observed in control and, to an even greater degree, in failing ventricular homogenates, represent a combination of these five phosphorylation sites or as yet novel serine or threonine residues, which become phosphorylated in ventricular failure. Indeed, other investigators (33) have documented that when the five putative sites are rendered nonphosphorylatable by mutation to alanines, other sites on the cTnI molecule become phosphorylated by protein kinase A and protein kinase C. We surmise that a similar phenomenon may also occur in experimental CHF in which prolonged and chronic activation of protein kinase signaling cascades phosphorylates both identified and novel cTnI sites blunting myofilament function and contractility in salient ways, which are functionally relevant, but are beyond the scope of investigation reported herein.

In closing, we have presented data which indicate that in two divergent animal models of CHF, LV myofilament function is depressed to a similar degree due, in part, to defects in the regulatory cTn complex. Furthermore, our data indicate that increased phosphorylation of cTnI as well as a shift of phosphates on cTnI likely plays a key role in reduced cTn functionality in experimental CHF. Future studies are needed to comprehensively examine and characterize which cTnI phospho-states are upregulated in heart failure and what functional effect phosphorylation of these sites has on myofilament function and in vivo cardiac contractility. Such knowledge is essential in elucidating specific molecular foci, which can be targeted for therapeutic interventions aimed at improving myofilament and ultimately ventricular function in the failing heart.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported, in part, by National Heart Lung and Blood Institute Grants R01-HL-64035, R01-HL-77195, RO1-HL-77195, PO1-HL-62426 (projects 1,4), T32-00888, and T32-007692 and by the American Heart Association Grants 0335199N and 0230038N. R. J. Belin was supported by a United Negro College Fund-MERCK Predoctoral Fellowship and American Physiological Society Porter Physiology Fellowship.


   ACKNOWLEDGMENTS
 
The authors thank Ed Allen for help with the troponin purification.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. P. de Tombe, Dept. of Physiology & Biophysics, Univ. of Illinois at Chicago, 835 S. Wolcott (M/C 901), Chicago, IL 60612 (email: pdetombe{at}uic.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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