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

Abstract

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...

Cardiac thin filament regulation

“…Although it is well recognized that disturbances in calcium regulation constitute an important mechanism in this process [7], it is now clear that heart failure is also associated with a decline in myofilament response to activator Ca 2+ [15,16,27,37,67,72]. Although isoform distribution and proteolytic cleavage of contractile proteins may play a role, (inappropriate) contractile protein phosphorylation has emerged as an important determinant of depressed myofilament function in various etiologies of heart failure [5,6,27,67]. For example, in experimental heart failure in the rat, we found a depression in myofilament function that was causally linked to alterations in Tn; a follow-up study demonstrated that this was due to, most likely, up-regulation of PKC-α activity and the subsequent phosphorylation of cardiac contractile proteins [5,6].…”

“…Although isoform distribution and proteolytic cleavage of contractile proteins may play a role, (inappropriate) contractile protein phosphorylation has emerged as an important determinant of depressed myofilament function in various etiologies of heart failure [5,6,27,67]. For example, in experimental heart failure in the rat, we found a depression in myofilament function that was causally linked to alterations in Tn; a follow-up study demonstrated that this was due to, most likely, up-regulation of PKC-α activity and the subsequent phosphorylation of cardiac contractile proteins [5,6]. Likewise, studies by van der Velden et al indicate that depressed myofilament function in human heart failure is associated with alterations in Tn phosphorylation, as well as other contractile proteins such as myosin light chain and myosin-binding protein C [27].…”

“…Recent investigations have provided new insights into the molecular mechanisms that underlie thin filament activation and the role of contractile protein phosphorylation in modulating myofilament function in the heart [37,67]. Moreover, contractile protein phosphorylation has emerged as an important determinant of depressed myofilament function in cardiac diseases such as the syndrome of congestive heart failure, cardiac hypertrophy, and diabetic cardiomyopathy [5,6,27,67]. Accordingly, this review is focused on the cardiac thin filament and its role in regulating cardiac myofilament dynamics both in health and disease.…”

The role of protein kinase C-mediated phosphorylation of sarcomeric proteins in the heart—detrimental or beneficial?

“…It is involved in numerous cellular processes, such as transcription of DNA, cell death, cell growth, Ca 2+ -handling, and also fine-tunes myofilament function. PKC isoforms have been implicated in several diseases, such as prostate and breast cancer, pulmonary disease, and heart failure (Belin et al 2007;Konopatskaya and Poole 2010;Parker 2003;Parker and Murray-Rust 2004;Sumandea et al 2004).…”

“…The regulatory role of PKC may be even larger during cardiac disease, as increased PKC activity and expression levels have been shown in the failing heart (Belin et al 2007;Braz et al 2004;Lamberts et al 2007). …”