The small GTPase RhoA plays an important role in transducing signals from the extracellular matrix and from a subset of G protein–coupled receptors, but its role in regulating cardiac physiology and pathophysiology has remained elusive. Early studies suggested that RhoA and its downstream effector ROCK contribute to development of cardiac hypertrophy (9
). We have observed, however, that while chronic expression of activated RhoA beginning in early development can induce hypertrophy (Supplemental Figure 1) and that a lethal dilated cardiomyopathy and heart failure develop with chronic high level expression of RhoA (7
), this does not occur in mice expressing low levels of RhoA in the adult heart. The CA-RhoA lines generated and examined in the current study show conditional (upon Dox removal) and modest (2- to 5-fold) increases in active GTP-RhoA, increases comparable to those induced in the mouse heart by treatment with agonists such as sphingosine-1-phosphate (13
) or by I/R (Figure ). Oxidative stress or inflammatory mediators released during ischemia and reperfusion would be expected to activate RhoA.Thus, our experimental model mimics and allows us to question the role that RhoA serves when the heart is initially exposed to I/R. Importantly, because RhoA is specifically expressed in cardiomyocytes in CA-RhoA mice and specifically deleted from cardiomyocytes in the RhoA-knockout mice, we can resolve the effects of RhoA in cardiomyocytes from its potential effects in other cell types in the heart. Our data clearly demonstrate that a modest level of RhoA activation in adult cardiomyocytes is not pathological but, remarkably, defines a previously unrecognized pathway for cardioprotection.
When CA-RhoA mouse hearts were challenged by I/R, we observed profound tolerance to both ex vivo and in vivo I/R injury. Protection was manifest as 60% to 70% reductions in infarct size, decreased LDH release, and markedly improved postischemic contractile performance. Parallel loss-of-function experiments using cardiac-specific RhoA-knockout mice demonstrated markedly increased infarct size and LDH release when cardiomyocytes did not express RhoA. These data provide substantial support for the conclusion that endogenous RhoA is activated in cardiomyocytes during I/R, where it functions to protect myocytes against I/R injury.
Further studies examined the mechanism by which RhoA mediates cardioprotection. In the CA-RhoA heart, the autophosphorylation of PKD was markedly increased, indicative of its activation. RhoA expression or RhoA activation in NRVMs also increased PKD phosphorylation. These data extend previously published work showing that PKD is activated when RhoA is heterologously expressed in COS-7 and HeLa cells (31
). There is also increased membrane distribution of PKCε and PKCδ in CA-RhoA hearts. These nPKC isozymes are known to phosphorylate and activate PKD (22
), and indeed we observed increased phosphorylation of established PKC sites on PKD in the CA-RhoA mouse heart.
There is, to our knowledge, no previous evidence that PKD mediates cardioprotection. Accordingly, we used inhibitors of PKD to test its role in the cardioprotection observed in the CA-RhoA mouse heart. Either of 2 classes of PKD inhibitor significantly reversed cardioprotection against I/R injury (Figure and Supplemental Figure 7). We further demonstrate that both RhoA and PKD are activated in response to oxidative stress induced by I/R in the WT mouse heart and that I/R injury in the WT mouse heart is exacerbated by inhibition of PKD. Finally, we provide evidence that the PKD activation that occurs with I/R is prevented when RhoA is genetically deleted from cardiomyocytes, and this is accompanied by increased infarct size and LDH release. Studies using NRVMs additionally show that H2O2 activates RhoA and PKD and that acute loss of function of RhoA or PKD by siRNA or pharmacological inhibitors increases H2O2-induced DNA fragmentation. Taken together, these data implicate RhoA and PKD in a previously unexplored protective signaling pathway to counteract the deleterious effects of oxidative stress on cardiomyocytes.
It is of note that RhoA and PKD, identified here as what we believe to be novel players in cardioprotection, could be involved in the pathways utilized by more established cardioprotective mediators. For example, nPKCs, particularly PKCε, mediate cardioprotection (33
). As a downstream target for phosphorylation by the nPKC isozymes (22
), PKD could contribute to their protective effects. The effects of the Rho-activated kinase, ROCK, in cardiac pathophysiology also bear comment, since several laboratories have reported salutary effects of inhibiting or deleting ROCK, suggesting that Rho activation in the heart is deleterious (5
). In this regard, a key element of the data presented here is that our models have allowed us to focus on what RhoA does in the cardiomyocyte, distinct from the potential ROCK-mediated functions of RhoA in fibroblasts (30
), arterial smooth muscle cells (37
), or endothelial cells (40
), where proliferation or membrane permeability changes would be maladaptive. The unexpected cardioprotective role of RhoA in cardiomyocytes, taken with evidence that it is activated at early times following reperfusion, suggests that RhoA is not always a villain and that one may want to facilitate rather than block its activation at early times during reperfusion of the ischemic heart.
In conclusion, our studies demonstrate that modest levels of RhoA activation in adult mouse cardiomyocytes provide striking protection against I/R injury and that genetic deletion of RhoA in cardiomyocytes renders the heart more susceptible to I/R damage. Our findings reveal a previously undescribed signaling pathway for RhoA in the heart and suggest that PKD mediates cardioprotection. These new insights into the effects of RhoA in I/R injury suggest that interventions that activate RhoA could play an important physiological or therapeutic role in promoting cardiomyocyte survival.