Induction of the matricellular protein CCN1 through RhoA and MRTF-A contributes to ischemic cardioprotection.

Activation of RhoA, a low molecular-weight G-protein, plays an important role in protecting the heart against ischemic stress. Studies using non-cardiac cells demonstrate that the expression and subsequent secretion of the matricellular protein CCN1 is induced by GPCR agonists that activate RhoA. In this study we determined whether and how CCN1 is induced by GPCR agonists in cardiomyocytes and examined the role of CCN1 in ischemic cardioprotection in cardiomyocytes and the isolated perfused heart. In neonatal rat ventricular myocytes (NRVMs), sphingosine 1-phosphate (S1P), lysophosphatidic acid (LPA) and endothelin-1 induced robust increases in CCN1 expression while phenylephrine, isoproterenol and carbachol had little or no effect. The ability of agonists to activate the small G-protein RhoA correlated with their ability to induce CCN1. CCN1 induction by S1P was blocked when RhoA function was inhibited with C3 exoenzyme or a pharmacological RhoA inhibitor. Conversely overexpression of RhoA was sufficient to induce CCN1 expression. To delineate the signals downstream of RhoA we tested the role of MRTF-A (MKL1), a co-activator of SRF, in S1P-mediated CCN1 expression. S1P increased the nuclear accumulation of MRTF-A and this was inhibited by the functional inactivation of RhoA. In addition, pharmacological inhibitors of MRTF-A or knockdown of MRTF-A significantly diminished S1P-mediated CCN1 expression, indicating a requirement for RhoA/MRTF-A signaling. We also present data indicating that CCN1 is secreted following agonist treatment and RhoA activation, and binds to cells where it can serve an autocrine function. To determine the functional significance of CCN1 expression and signaling, simulated ischemia/reperfusion (sI/R)-induced apoptosis was assessed in NRVMs. The ability of S1P to protect against sI/R was significantly reduced by the inhibition of RhoA, ROCK or MRTF-A or by CCN1 knockdown. We also demonstrate that ischemia/reperfusion induces CCN1 expression in the isolated perfused heart and that this functions as a cardioprotective mechanism, evidenced by the significant increase in infarct development in response to I/R in the cardiac specific CCN1 KO relative to control mice. Our findings implicate CCN1 as a mediator of cardioprotection induced by GPCR agonists that activate RhoA/MRTF-A signaling.

PLCε, PKD1, and SSH1L transduce RhoA signaling to protect mitochondria from oxidative stress in the heart.

Activation of the small guanosine triphosphatase RhoA can promote cell survival in cultured cardiomyocytes and in the heart. We showed that the circulating lysophospholipid sphingosine 1-phosphate (S1P), a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) agonist, signaled through RhoA and phospholipase Cε (PLCε) to increase the phosphorylation and activation of protein kinase D1 (PKD1). Genetic deletion of either PKD1 or its upstream regulator PLCε inhibited S1P-mediated cardioprotection against ischemia/reperfusion injury. Cardioprotection involved PKD1-mediated phosphorylation and inhibition of the cofilin phosphatase Slingshot 1L (SSH1L). Cofilin 2 translocates to mitochondria in response to oxidative stress or ischemia/reperfusion injury, and both S1P pretreatment and SSH1L knockdown attenuated translocation of cofilin 2 to mitochondria. Cofilin 2 associates with the proapoptotic protein Bax, and the mitochondrial translocation of Bax in response to oxidative stress was also attenuated by S1P treatment in isolated hearts or by knockdown of SSH1L or cofilin 2 in cardiomyocytes. Furthermore, SSH1L knockdown, like S1P treatment, increased cardiomyocyte survival and preserved mitochondrial integrity after oxidative stress. These findings reveal a pathway initiated by GPCR agonist-induced RhoA activation, in which PLCε signals to PKD1-mediated phosphorylation of cytoskeletal proteins to prevent the mitochondrial translocation and proapoptotic function of cofilin 2 and Bax and thereby promote cell survival.

Functional role of anion channels in cardiac diseases.

In comparison to cation (K+, Na+, and Ca2+) channels, much less is currently known about the functional role of anion (Cl-) channels in cardiovascular physiology and pathophysiology. Over the past 15 years, various types of Cl- currents have been recorded in cardiac cells from different species including humans. All cardiac Cl- channels described to date may be encoded by five different Cl- channel genes: the PKA- and PKC-activated cystic fibrosis tansmembrane conductance regulator (CFTR), the volume-regulated ClC-2 and ClC-3, and the Ca2+-activated CLCA or Bestrophin. Recent studies using multiple approaches to examine the functional role of Cl- channels in the context of health and disease have demonstrated that Cl- channels might contribute to: 1) arrhythmogenesis in myocardial injury; 2) cardiac ischemic preconditioning; and 3) the adaptive remodeling of the heart during myocardial hypertrophy and heart failure. Therefore, anion channels represent very attractive novel targets for therapeutic approaches to the treatment of heart diseases. Recent evidence suggests that Cl- channels, like cation channels, might function as a multiprotein complex or functional module. In the post-genome era, the emergence of functional proteomics has necessitated a new paradigm shift to the structural and functional assessment of integrated Cl- channel multiprotein complexes in the heart, which could provide new insight into our understanding of the underlying mechanisms responsible for heart disease and protection.