Protein regulation in heart and skeletal muscle

We are studying cardiac remodeling and inflammation-dependent skeletal muscle atrophy. We are particularly interested in the ubiquitin proteasome system (UPS). We showed earlier that muscle RING finger (MuRF) 1 and MuRF3 are important for maintaining cardiac and skeletal muscle structure and function. We are currently identifying and characterizing the regulation and function of MuRF proteins in myocytes. We identified the stress responsive serine/threonine kinase protein kinase D1 (PKD1) as an important regulator of pathological cardiac remodeling and fiber type specification in skeletal muscle. We investigated PKD1-dependent control of the class IIa histone deacetylase/myocyte enhancer factor-2 axis in chronic cardiac stress situations, and work on non-transcriptional functions of PKD1 in heart and skeletal muscle.

 

Protein quality control and degradation

The adult heart responds to biomechanical stress and neurohormonal signaling by hypertrophic growth, accompanied by fibrosis, and activation of a fetal gene program leading to a diminished cardiac function. Class II histone deacetylases (HDACs) are negative regulators of pathological cardiac remodeling via the myocyte enhancer factor-2 (MEF2) transcription factor, an activator of heart disease. Protein kinase D1 (PKD1) is a stress-responsive kinase that phosphorylates class II HDACs, resulting in their dissociation from MEF2 with subsequent activation of MEF2 target genes. Cardiac PKD1 is activated in response to arterial hypertension, pressure overload, and chronic neurohormonal activation. We generated mice with a conditional PKD1-null allele. Mice with cardiac-specific deletion of PKD1 showed diminished cardiac hypertrophy, interstitial and perivascular fibrosis, and fetal gene activation as well as improved cardiac function in response to chronic pressure overload or chronic angiotensin II and adrenergic signaling. These findings demonstrate that PKD1 in cardiomyocytes plays a key role in mediating stress-dependent remodeling and reprogramming of gene expression in the adult heart (Figure 1). However, in addition to transcriptional regulation PKD1 regulates myocyte contractility by phosphorylating contractile proteins. In further projects, we focus on non-transcriptional functions of PKD1 in acute cardiac stress response.

 

PKD1 signaling promotes slow-twitch skeletal-muscle fibers

Skeletal muscle is heterogeneous. Based on the expression of myosin heavy chain (MyHC) isoforms, myofibers can be classified as type I (slow) or type II (fast) fibers; type II fibers are further categorized into type IIa, type IId/x, and type IIb. The MEF2 transcription factors regulate skeletal muscle remodeling and fiber type specification. In adult skeletal muscle, the activity of MEF2 is tightly regulated through association with class II HDACs.  We found that PKD1 is preferentially expressed in type I myofibers. Forced expression of constitutively active PKD1 (caPKD1) in type II fibers of transgenic mice potently stimulated the transcriptional activity of MEF2, and in turn promoted their conversion toward a slow myofiber phenotype. Consistent with this fiber type switch, skeletal muscles derived from caPKD1 transgenic mice were resistant to fatigue during repetitive contractions. In contrast, skeletal muscle-specific deletion of PKD1, by use of a conditional PKD1 null allele, increased the susceptibility to fatigue of type I soleus muscle, but not that of type II extensor digitorum longus muscle. Our data showed that PKD1 signaling plays an important role in the control of skeletal muscle fiber type via its stimulatory activity with respect to MEF2 and that PKD1 modulates skeletal muscle function and phenotype.

 

Muscle RING-finger 1 and 3 (MuRF) proteins

Striated muscle structure and function is maintained by precise control of protein synthesis and degradation; abnormalities in these processes can give rise to myopathies. The UPS degrades misfolded proteins. UPS substrate specificity is mediated by E3 ubiquitin ligases, such as RING-finger proteins. Muscle RING-finger (MuRF) proteins 1, 2, and 3 comprise a subfamily of the RING-finger E3 ubiquitin ligases that are specifically expressed in the heart and skeletal muscle. We generated MuRF3 knockout mice and demonstrated that MuRF3 is involved in maintaining cardiac structure and function, and the integrity of the ventricular wall following acute myocardial infarction. Additionally, we found that MuRF1–/–MuRF3–/– double mutant (DKO) mice display a distinct cardiac and skeletal muscle myopathy reminiscent of myosin storage myopathy (protein surplus myopathy) in humans. Cardiac and skeletal muscles of DKO mice displayed a striking subsarcolemmal accumulation of MyHC accompanied by cardiac hypertrophy, decreased cardiac function and reduced maximal force development of the skeletal muscle. MuRF1 and MuRF3 interact specifically with β/slow MyHC and MyHCIIa and utilize UbcH5a, -b, and -c as E2 ubiquitin–conjugating enzymes to catalyze the ubiquitinylation and degradation of these proteins. We are now working on the regulation of function and activity of MuRF proteins.

 

Inflammation induced skeletal muscle atrophy in critically ill patients

Intensive care unit (ICU) acquired skeletal muscle weakness syndrome (ICUAW) is common in critically ill patients; the consequences are devastating and the costs are enormous. The causes are largely unknown. We found that atrophy of type II, but not type I, skeletal myofibers commonly occurs in severely affected ICU patients. Bona fide atrophy related proteins (atrogens) were highly induced in ICUAW early in the disease process. Interestingly, changes in gene expression of atrogens were correlated with the degree of type II myofiber atrophy. Finally, microarray analysis showed disease specific regulation of gene expression depending on the severity of ICUAW. We believe that UPS dependent protein degradation and transcriptional regulation are involved in the disease course of ICUAW. Further analyses will focus on these pathways to better understand the molecular mechanisms of ICUAW.

Figure 1. We show the effects of cardiomyocyte specific PKD1 elimination. After angiotensin II (Ang II) treatment, PKD1 mutant mice (cKO) displayed less cardiac hypertrophy and a reduction in perivascular and interstitial fibrosis (hematoxylin and eosin and Masson's trichrome stainings; top panel).  Atrial natriuretic factor (ANF), ß-myosin heavy chain (β-MHC), and procollagen, type I, α2 (Col1α2) expression was also compromised in PKD1 mutant mice (cKO) (bottom panel).